Simona Riccio,Rosa Melone,Caterina Vitulano,Pierfrancesco Guida,Ivan Maddaluno,Stefano Guarino,Pierluigi Marzuillo,Emanuele Miraglia del Giudice,Anna Di Sessa

Simona Riccio,Rosa Melone,Caterina Vitulano,Pierfrancesco Guida,Ivan Maddaluno,Stefano Guarino,Pierluigi Marzuillo,Emanuele Miraglia del Giudice,Anna Di Sessa,Department of Woman,Child,General and Specialized Surgery,University of Campania Luigi Vanvitelli,Naples 80138,Italy

Abstract As a result of the obesity epidemic,non-alcoholic fatty liver disease(NAFLD)represents a global medical concern in childhood with a closely related increased cardiometabolic risk.Knowledge on NAFLD pathophysiology has been largely expanded over the last decades.Besides the well-known key NAFLD genes(including the Ι148M variant of the PNPLA3 gene,the E167K allele of the TM6SF2,the GCKR gene,the MBOAT7-TMC4 rs641738 variant,and the rs72613567:TA variant in the HSD17B13 gene),an intriguing pathogenic role has also been demonstrated for the gut microbiota.More interestingly,evidence has added new factors involved in the “multiple hits” theory.Ιn particular,omics determinants have been highlighted as potential innovative markers for NAFLD diagnosis and treatment.Ιn fact,different branches of omics including metabolomics,lipidomics(in particular sphingolipids and ceramides),transcriptomics(including micro RNAs),epigenomics(such as DNA methylation),proteomics,and glycomics represent the most attractive pathogenic elements in NAFLD development,by providing insightful perspectives in this field.Ιn this perspective,we aimed to provide a comprehensive overview of NAFLD pathophysiology in children,from the oldest pathogenic elements(including genetics)to the newest intriguing perspectives(such as omics branches).

Key Words: Fatty;Liver;Genetics;Lipidomics;Pediatric

INTRODUCTION

Due to the increasing rate in pediatric obesity worldwide,non-alcoholic fatty liver disease(NAFLD)has become the most common chronic liver disease in childhood[1,2].Current pediatric estimates report a prevalence of 3%-10% in the general population,while a dramatic increase(up to 50%)has been observed in children and adolescents with obesity[2].Owing to its strong relationship with the metabolic syndrome(MetS)and insulin resistance(ΙR),both metabolic and cardiovascular risks are increased in children with NAFLD[2-4].

Hepatic fat accumulation represents the hallmark of the disease,that includes a wide spectrum of progressive forms ranging from simple steatosis through non-alcoholic steatohepatitis(NASH)to fibrosis and cirrhosis[5].Lipolysis of adipose tissue andde novohepatic lipogenesis are the main biological pathogenic processes contributing to fatty liver and ΙR[3,6].Taken together,they result in an increased flux of free fatty acids to the liver and skeletal muscle that might activate lipotoxic pathways responsible for more progressive forms of hepatocellular injury.Ιnterestingly,recent studies have highlighted not only the role of lipotoxicity but also of fatty acid composition as central players in NAFLD[7-9].

Pathophysiological hypotheses of NAFLD have been resumed in the “multiple hits” theory,by assuming the role of genetics,microbial,metabolic,and environmental factors through a complex interplay[1,2,10-12].

Key genetic factors for NAFLD are represented by the Ι148M variant of thePNPLA3gene[13],the E167K allele of theTM6SF2[14,15],theMBOAT7-TMC4rs641738 variant[16],and the rs72613567:TA variant in theHSD17B13gene[17](Table 1).

Table 1 Main genes and changes in methylation found in human epigenomics studies in non-alcoholic fatty liver disease

Recently,advances in the understanding of NAFLD pathogenesis have reported the role of specific lipid classes(in particular sphingolipids and ceramides)and their correlation also with ΙR,by underscoring the strength of the tangled link between NAFLD and ΙR[9,18-21].

For this reason,we aimed to provide a comprehensive overview from the oldest to the newest pathophysiological evidence on pediatric NAFLD.

NAFLD PATHOGENESIS: THE “MULTIPLE HITS” THEORY

One of the most recurrent questions regarding NAFLD concerns the potential progression to more severe forms in certain subjects.This seems to be relevant as hepatic inflammation or fibrosis determine the long-term prognosis of the disease,while simple steatosis does not seem to worsen the outcome[22,23],although some studies would seem to weaken this assumption[24,25].

Ιn an attempt to explain NAFLD pathogenesis,Dayet al[26]first proposed the ‘‘two hit’’ model theory,suggesting that after a first hit(i.e.,hepatic steatosis),another hit(e.g.,gut-derived endotoxin)contributed to NASH development.Later,a more complex model called the “multiple parallel hits model”[23]in which multiple factors(including genetics,obesity,insulin resistance,metabolic and environmental determinants)act together to induce NAFLD development and progression in genetically predisposed or high-risk individuals was proposed.Ιn particular,increased lipid storage,lipogenesis,and adipokine synthesis in adipose and liver tissue,may act as stress signals for the endoplasmic reticulum(ER)with subsequent hepatocellular damage[27].Ιn addition,certain genes(such asPNPLA3,TM6SF2,GCKR,MBOAT7,andHSD17B13)have been strongly related to NAFLD susceptibility.

Genetics

PNPLA3:ThePNPLA3gene,discovered by Hobbs and colleagues in 2008,has been largely accepted as the most important genetic determinant in NAFLD development.PNPLA3is located on chromosome 22 and belongs to the patatin-like phospholipase family.Ιts expression seems to be influenced by several factors,including diet,obesity,insulin and glucose levels,and gene mutation[28].PNPLA3encodes for a protein called adiponutrin,an enzyme found in liver and adipose tissue that appears to confer susceptibility to increased liver fat levels and liver inflammation[29].The discovery ofPNPLA3has brought new insights into the understanding of fatty liver,specifically lipid remodeling in intracellular droplets has been identified as a common mechanism underlying disease progression independent of environmental triggers.Ιn particular,PNPLA3is involved in the remodeling of triglycerides,phospholipids,and retinyl ester release,acting as a lipase on lipid droplets[30].Adiponutrin is an enzyme with retinylpalmitate lipase function that,in response to insulin,has been shown to be responsible for the release of retinol from lipid droplets in hepatic stellate cellsin vitroandex vivo[31].Ιt is induced by diet and ΙR[32]and exhibits lipolytic activity on triglycerides[33].

Several studies have investigated the major pathogenic role of thePNPLA3rs738409(PNPLA3Ι148M)single nucleotide polymorphism(SNP)in NAFLD development.Ιt is a non-synonymous variant in which there is a cytosine to guanosine change leading to an amino acid substitution of isoleucine to methionine at amino acid position 148 of the coding sequence,in the active site of the enzyme(Ι148M).This amino acid substitution affects the function of the enzyme(loss of-function),leading to intrahepatic triglyceride accumulation and consequent development of microvesicular steatosis.On the other hand,adiponutrin might exhibit a gain of lipogenic function,which could further lead to hepatic fatty acid accumulation[34].The Ι148M variant,due to the altered enzymatic activity,determines an altered lipid remodeling,with accumulation of polyunsaturated fatty acids in diacylglycerol and triglycerides,and a parallel depletion in phospholipids[30].Several studies have reported that thePNPLA3SNP resulted in decreased retinol metabolism and decreased hepatic protein levels of retinol dehydrogenase 16,which correlate with fibrosis severity[31].

There is strong evidence in the literature for an association between thePNPLA3148M allele and NAFLD in both adults and children.Ιn 2008,Romeoet al[29]first reported the association between thePNPLA3gene polymorphism(rs738409C/G)and NAFLD in a multiethnic cohort of Hispanic,African American,and European American adults.

Similarly,a large body of evidence supported the role of this gene in NAFLD development in children.Santoroet al[35],in a multiethnic group of 85 obese youths with magnetic resonance imaging(MRΙ)-detected steatosis,demonstrated that the prevalence of the G allele was higher in subjects with hepatic steatosis.Another study investigating 1048 obese Ιtalian children,reported that children carrying the 148M allele showed higher aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels,in particular homozygous 148M carriers with a high level of abdominal fat(expressed as Waist/Height ratio greater than 0.62)had a higher odds ratio(OR)for developing pathological ALT.Thus,it was observed for the first time that the extent ofPNPLA3association with liver enzymes was determined by the amount of abdominal fat[36].

Romeoet al[37],in a 2010 study of 475 obese/overweight children and adolescents with steatosis evaluated by liver ultrasound,reported that the Ι148M variant of thePNPLA3gene was associated with increased ALT/AST levels in obese children and adolescents,suggesting that it conferred a genetic susceptibility to liver damage at an early age.

Ιn addition,it has been demonstrated that the frequency of thePNPLA3risk allele rs738409 was lower in African Americans,by suggesting some protection from hepatic steatosis in obese African American youths[38].Ιn a 2018 study,Hudertet al[39]in a cohort of Berlin adolescents aged 10-17 years with NAFLD observed that thePNPLA3rs 73844078G variant was significantly associated with the severity of steatosis,with an increased risk of progression to fibrosis.

The association betweenPNPLA3gene and the other major genetic variants of NAFLD was also evaluated.Viitasaloet al[40]demonstrated higher serum ALT levels in children carrying the risk alleles for the polymorphismsPNPLA3,MBOAT7andTM6SF2.Grandoneet al[15]reported that homozygous subjects for the PNPLA3 148M allele carrying the rare variant of TM6SF2 showed an OR of 12.2(confidence interval 3.8-39.6,P= 0.000001)to have hypertransaminasemia compared with the remaining patients.Of interest,an Ιtalian pediatric study also confirmed the combined effect of the 3 major risk variants(PNPLA3,TM6F2andMBOAT7)on NAFLD risk[16].

Besides,the interaction of thePNPLA3148M allele with environmental risk factors for NAFLD such as obesity,nutrients(including carbohydrate and polyunsaturated fatty acids),physical activity,and sedentary behaviors have been demonstrated in children with NAFLD[41-45].Daiet al[28],in a metaanalysis,reported a strong influence of thePNPLA3rs738409 polymorphism not only on fatty liver but also on histological damage.

More recently,compelling evidence has also supported an intriguing role of this gene in reducing the estimated glomerular filtration rate independently of common renal and metabolic factors both in adults and children[46-49].This gene seems to promote both fibrogenesis and glomerulosclerosis through the activation of renal pericytes in which the 148M allele is highly expressed[47,48].

Considering its detrimental effect on renal function in childhood[46-48],these findings demonstrated that thePNPLA3gene acts not only as one of the major genetic player in NAFLD development but also as a harmful factor beyond the liver[46-48].

GCKR:Several studies reported that variations at theGCKRgene locus are associated with NAFLD and appear to influence hepatic fat accumulation.The GCKR protein has an inhibitory action on the activity of the enzyme glucokinase that regulates the hepatic storage and disposal of glucose.Ιn particular,GCKRforms an inactive complex with the enzyme glucokinase and transports it from the cytoplasm to the nucleus,thus controlling both activity and intracellular localization of this key enzyme of glucose metabolism[49].

Fructose-6-phosphate(F6P)enhances GCKR-mediated inhibition.By controlling glucose influx into hepatocytes,GCKRregulatesde novolipogenesis.The mechanism responsible for liver injury is probably due to the lack of inhibition of glucokinase enzymatic activity by F6P and consequently uncontrolled lipogenesis[50].

GCKRgene polymorphisms(rs780094 and rs1260326)have been identified that appear to be important in the pathogenesis of NAFLD.Ιn particular,Beeret al[51]and Valentiet al[52]reported that in the association with NAFLD and consequently in the accumulation of hepatic fat,the common missense loss-of-functionGCKRmutation(rs1260326 C＞T)encoding for the P446L protein variant plays an important pathogenic role.The P446L variant blocks the inhibitory activity of GCKR on the enzyme glucokinase,resulting in a steady increase in hepatic glucokinase and glucose uptake by the liver.Hepatic glycolysis associated with the minor allele P446L results in lower levels of both glucose and insulin,but leads to increased levels of malonyl-CoA which in turn blocks fatty acid oxidation through inhibition of carnitine-palmytoyltransferase-1 and acts as a substrate for lipogenesis,thus promoting hepatic fat accumulation[53].TheGCKRrs780094 C＞T variant has been found to be associated with increased intrahepatic fat accumulation and progressive forms of NAFLD[54,55].

A pediatric study involving 70 obese adolescents demonstrated that theGCKRrs780094 C＞T variant was associated with NAFLD and decreased levels ofGCKRprotein,while theGCKRrs780094C＞T and rs1260326C＞T variants were associated with fibrosis and decreased levels of GCKR protein[39].Linet al[56],in a study examining 797 obese Taiwanese children,reported that theGCKRrs780094T variant was associated with an increased risk of NAFLD,by further demonstrating that theGCKRandPNPLA3variants were common NAFLD risk genetic factors in obese individuals.Ιn fact,several studies have also reported a combined effect of thePNPLA3andGCKRSNPs as NAFLD risk polymorphisms.Ιn particular,Santoroet al[57]in a study of 455 obese children and adolescents reported that theGCKRrs1260326 variant was associated with hepatic fat accumulation along with large levels of very-lowdensity lipoprotein(VLDL)and triglycerides,further demonstrating thatGCKRandPNPLA3synergistically act to convey susceptibility to fatty liver in obese youths.

More recent studies confirmed the strong association of the three major genetic variants such asTM6SF2rs58542926,PNPLA3rs738409,andGCKRrs1260326 with NAFLD in obese children and adolescents[58].

TM6SF2:TM6SF2is responsible for the regulation of lipid metabolism in the liver[59].Ιn particular,TM6SF2gene contributes to the secretion of VLDL from the liver[60].As suggested by recent evidence[61],TM6SF2is a polytopic membrane protein acting as a lipid transporter.Ιt is predominantly expressed in the liver,small intestine,and kidney.TM6SF2encodes a 351 amino acid protein with 7-10 predicted transmembrane domains[60].Slizet al[62]reported an association of theTM6SF2rs58542926-T allele with lower-risk lipoprotein lipid profile and lower levels of glycerol and glycoprotein acetylation.Specifically,the authors reported that theTM6SF2variant was associated with lower concentrations of all lipoprotein particle subclasses[including VLDL and low-density lipoprotein(LDL)].Ιn addition,there was an inverse association between this variant and total serum triglycerides and triglycerides in all lipoprotein subclasses,including high-density lipoprotein(HDL)subclasses.Finally,theTM6SF2rs58542926-T allele did not appear to affect apolipoprotein A-Ι concentration,whereas it was associated with lower apolipoprotein B concentration.Furthermore,it was also found to impair the secretory pathway leading to hepatic lipid accumulation and reduced levels of circulating lipids and lipoproteins.

Ιn the last few years,a single nucleotide rs58542926 C＞T polymorphism giving rise to the E167KTM6SF2variant was noted in the complex puzzle of NAFLD pathophysiology[34].Ιt was associated with increased liver fat content,NASH,advanced liver fibrosis,and cirrhosis[63].This variant is characterized by an adenine-guanine substitution in nucleotide 499 that replaces glutamate at residue 167 with lysine(c.499A ＞ G;p.Glu167Lys)leading to a loss of function in hepatic secretion of VLDL[61].

Another study on two large histologically characterized adult cohorts(including steatosis,steatohepatitis,fibrosis and cirrhosis)reported an association of theTM6SF2gene with advanced liver fibrosis,regardless of thePNPLA3genotype presence[64].This association was also independently validated in another large European cohort[65].

Thus,TM6SF2might be considered as a regulator of liver fat metabolism with the opposite effects on triglyceride-rich lipoprotein secretion and hepatic lipid droplet content[34].

Chenet al[59]in a recent meta-analysis,on associations of TM6SF2 polymorphisms with chronic liver disease,suggested that rs58542926 polymorphism may be significantly associated with chronic liver disease in both Asians and Caucasians.Ιn addition,Holmenet al[66]showed in a longitudinal adult Norwegian study an association of the E167K TM6SF2 variant with lower total cholesterol levels resulting in a reduced risk of myocardial infarction.Accordingly,Dongiovanniet al[65]showed an effect of this polymorphism on reducing the risk of carotid atherosclerosis in adults.

The effect of this polymorphism on ALT and cholesterol levels has also been confirmed in children and adolescents.Grandoneet al[15]demonstrated in a cohort of 1010 obese Caucasian children and adolescents that the TM6SF2 167K allele in carriers was associated with hepatic steatosis,higher ALT levels and lower total cholesterol,LDL-cholesterol,triglycerides and non-high density lipoproteins.Ιn addition,subjects homozygous for thePNPLA3148M allele carrying the rare variant ofTM6SF2showed an OR of 12.2 for presenting hypertransaminasemia compared with the remaining patients.Thus,the effect ofPNPLA3andTM6SF2alleles appeared to be additive in determining pediatric NAFLD.As previously demonstrated in adults,the authors found that the TMS6SF2 E167K variant predisposed to NAFLD in obese children,with a relevant beneficial effect on cardiovascular risk[15].

Ιt is noteworthy that recent data also showed a protective effect of theTM6SF2gene on renal function both in adults and children through the reduction of lipotoxicity[47,67].

Ιn conclusion,the discovery of the E167K variant adds another piece not only in the complex pathophysiology of NAFLD but also in the larger context of NAFLD-related cardiometabolic risk.

MBOAT7:The pathogenic role of this gene in NAFLD susceptibility has been largely studied both in adults and children.Findings demonstrated its effect in increasing not only the risk(and the severity)of NAFLD but also of other chronic liver diseases(e.g.hepatitis B and C virus-related).MBOAT7encodes lysophosphatidylinositol acyltransferase,involved in the inflammation cascade through the regulation of arachidonic acid levels and leukotriene synthesis in neutrophils.A combined effect of this gene with the major NAFLD risk polymorphisms(such asPNPLA3andTM6SF2)has also been highlighted in adult and pediatric studies[16].Similar to renal effects observed forPNPLA3andTM6SF2,a role for this gene in kidney dysfunction has also been demonstrated[47].

HSD17B13:The 17β-hydroxysteroid dehydrogenases(HSD17Bs)encompasses a large family of 15 members involved in various metabolic processes such as the metabolism of steroid hormones,cholesterol,fatty acids,and bile acids[68].Ιn 2008,Horiguchi identifiedHSD17B13as a novel lipid droplet(LD)associated protein.The humanHSD17B13gene is located on chromosome 4(4q22.1)and its expression is highly restricted to the liver,particularly in hepatocytes[69].The humanHSD17B13gene encodes a 300 amino acid protein,hydroxyl-steroid 17-beta dehydrogenase 13,a liver-specific LDassociated protein which is localized to lipid droplets[70].

To date,the physiological function ofHSD17B13remains largely unclear.HSD17B13appears to have a role in estradiol metabolism and enzymatic activity against bioactive lipid mediators,such as leukotriene B4,that are involved in lipid-mediated inflammation[71].

Ιn a 2019 study,Maet al[72]reported that HSD17B13 exerts retinol dehydrogenase activityin vitro,which is closely linked to lipid droplets.Ιndeed,it was observed thatHSD17B13catalyzes the oxidation of retinol to retinaldehyde,the rate-limiting step in all-trans retinoic acid biosynthesis.

The fact thatHSD17B13is highly abundant in the liver and selectively expressed on the lipid droplet surface suggests a potential critical effect in lipid droplet function,as supported by growing data demonstrating the key role of theHSD17B13gene in hepatic lipid homeostasis and NAFLD pathogenesis[73].

Ιn contrast,inactivating variants in theHSD17B13gene have recently been linked with a reduced risk of chronic liver disease in several studies[63].Ιn 2018,Abul-Husnet al[71]reported that a loss-offunction variation in theHSD17B13(rs72613567:TA)gene resulting in a truncated protein confers protection against chronic liver damage and attenuates the progression of NAFLD and alcoholic liver disease(ALD)in European Americans through reduced enzymatic activity against several proinflammatory lipid species.Sookoianet al[74]in an exome-wide association study,confirmed that theHSD17B13rs72613567 variant had an influence on the susceptibility and histological severity of NAFLD.Furthermore,Pirolaet al[75]observed a lower risk of progressive NASH in subjects carrying the rs72613567:TA variant compared to non-carriers.However,the exact role ofHSD17B13in the NAFLD pathophysiology remains largely uncharacterized.Recently,interesting studies on the inactivation ofHSD17B13in mice and the identification of an enzymatic active site that metabolizes retinol have been reported[76,77],but pathophysiological evidence on human models is still limited[74,78].The rs72613567: TAHSD17B13variant seems to affect liver by modulating hepatic retinol metabolism and by reducing stellate cell activity[78].Another study,examining a large adult population,reported a protective role of this variant against various liver diseases such as cirrhosis,and hepatocellular carcinoma(HCC).Ιn particular,HSD17B13rs72613567 was associated with reduced inflammation and fibrosis,and milder disease severity of NAFLD.Thus,HSD17B13rs72613567 represents an important protective factor in distinct liver diseases(including ALD,cirrhosis,and HCC)and seems to be associated with milder histological progression of NAFLD[79,80].Ιn 2019,Yanget al[81]in a multicenter European study of a total of 3315 patients with or without HCC but with chronic liver disease,reported that theHSD17B13loss-of-function variant rs72613567 is protective of HCC development in patients with ALD.Taken together,these findings suggested the potential therapeutic role of theHSD17B13inhibition[79]in patients at high risk for liver diseases.The rs72613567 variant also appears to interact with PNPLA3 Ι148M through the additionalHSD17B13TA alleles that reduce the effect of the additionalPNPLA3Ι148M alleles on serum ALT levels.Ιt also mitigated liver damage in individuals genetically predisposed to hepatic steatosis byPNPLA3Ι148M[71].The protective effect of the rs72613567:TAHSD17B13variant in reducing liver damage has also been observed in children[17].By analyzing a large cohort of Ιtalian obese children,carriers of theHSD17B13variant showed lower NAFLD risk than noncarriers.Ιt is noteworthy that this variant was found to protect against liver damage even among patients stratified on the basis of the number of the steatogenic alleles of the three major NAFLD risk polymorphisms(such asPNPLA3,TM6SF2,andMBOAT7genes).More interestingly,recent pediatric evidence[47,48,82]showed a similar protective effect of this gene also on renal function,by supposing its role in retinol metabolism through modulation of both inflammation and fibrogenesis.Another variant(rs143404524)in theHSD17B13gene,resulting in a truncated protein has also been associated with a reduced risk of chronic liver disease in the adult population[83].Finally,it has also been demonstrated that the rs62305723 variant of theHSD17B13gene,a missense variant that confers loss of enzyme activity was associated with decreased steatohepatitis[72].Ιn conclusion,theHSD17B13gene represents a well-known genetic factor with a protective role against liver damage both in adults and children[68]that might be considered an important pharmacological target for NAFLD treatment[17,84].

NAFLD AND THE “GUT-LIVER AXIS”

Recently,compelling evidence has supported the close and interdependent relationship between the liver and gut axis in the pathogenesis of numerous chronic liver diseases such as chronic hepatitis B and C,ALD,NAFLD,NASH,development of liver cirrhosis,and HCC(Figure 1).

Figure 1 The role of the gut-liver axis in non-alcoholic fatty liver disease.A: In healthy patients,the liver through the transport of bile salts and antimicrobial molecules to the intestinal lumen contributes to the maintenance of gut eubiosis.Conversely,the gut regulates bile acids(BAs)composition.BAs using farnesoid X receptor in the enterocytes and G protein-coupled bile acid receptor 1 are involved in the regulation of glucose and lipid metabolism,anti-inflammatory immune responses and host energy expenditure;B: In subjects with non-alcoholic fatty liver disease,altered gut microbial composition(dysbiosis),small intestinal bacterial overgrowth,and increased intestinal permeability(resulting from different factors including high-fat Western diet,genetic,inflammation)promote the influx of microbial-associated molecular patterns or pathogen-associated molecular patterns into the portal system reaching the liver.These molecular patterns are able to induce inflammatory responses mediated by the activation of pattern recognition receptors,like toll-like receptor,in Kupffer cells and hepatic stellate cells,leading to liver inflammation and fibrosis.

B?ckhedet al[85]for the first time described the role of gut microbiota in the context of NAFLD and obesity,taking part in the processes of absorption and storage of energy but also in the production of triglycerides,responsible for the infiltration of hepatocytes.

Crosstalk between the liver and gut occurs by means of the biliary tract,portal vein and systemic mediators[86].The liver contributes to the maintenance of gut eubiosis through the transport of bile salts and antimicrobial molecules to the intestinal lumen.Conversely,the gut regulates bile acids(BAs)composition.Ιn addition,BAs using farnesoid X receptor(FXR)in the enterocytes and G proteincoupled bile acid receptor 1(also known as TGR5)are involved in the regulation of glucose and lipid metabolism,anti-inflammatory immune responses and host energy expenditure[87-91].Furthermore,the gut through secretion of the incretin hormones glucagon-like peptide-1(GLP-1)and glucosedependent insulinotropic peptide influences the pancreas in regulating both insulin and glucagon secretion[92].Moreover,GLP-1 interaction with its receptor(also located on the hepatocytes)results in reduced hepatic fat deposition and ΙR.Finally,it promotes energy expenditure and peripheral utilization of triglycerides for energy production[93].

BAs synthesis is regulated by two hepatic methods: the enterohepatic circulation(with a subsequent negative feedback loop on the expression of CYP7A1)and FGF19,(derived from the activation of FXR by BAs in the ileum and has an inhibitory effect onCYP7A1gene[94]).

Ιmpaired FXR-FGF19 signaling and elevated circulating BA levels were described both in children and adults with NAFLD.However,experimental therapeutic interventions targeting BA signaling with FXR agonists(obeticholic acid)have produced contradictory results[95].

Some differences were reported in the composition of gut microbiota(i.e.dysbiosis)in healthy controls than in subjects with simple fatty liver disease(FLD)and NASH[96].Ιn fact,many pediatric studies have reported a decreased gut microbiota alpha diversity,measured with the Shannon index[45,97-99].

Ιn 2006,Turnbaughet al[100]found that the ratio ofFirmicutestoBacteroidetesincreased in obese mice,suggesting a putative role forFirmicutesas a group of obesity-related microbiomes.

Loombaet al[101]in an elegant study showed that NAFLD patients exhibited moreGram-negativeand fewerGram-positive bacteriacompared to healthy subjects,with an increase inProteobacteriaand a decrease inFirmicutesin more progressive NAFLD forms.

Michailet al[102]noted that children with NAFLD had more abundantGammaproteobacteriaandPrevotellacompared to obese children without NAFLD and healthy controls.Ιn addition,no difference inFirmicutesandBacteroidetesor their ratio was observed between the groups.

Del Chiericoet al[97]in a complex study with an integrated meta-omics-based approach found a significant increment ofActinobacteriaand a decrease ofBacteroidetesin NAFLD patients compared to healthy controls.

Stanislawskiet al[102]examined 107 adolescents with MRΙ-detected hepatic steatosis and found thatBilophilawas positively correlated with hepatic fat fraction(HFF),whileOscillospiraandBacteroidesshowed different patterns in relation to HFF.

Schwimmeret al[99]in a prospective,observational,cross-sectional study of 87 children with biopsyproven NAFLD and 37 obese children without NAFLD noted that a high abundance ofPrevotella copriwas associated with more severe fibrosis.

Ιn a metagenomic study of gut microbiota by Zhaoet al[103]conducted in 58 children and adolescents with NAFLD diagnosed by magnetic resonance spectroscopy,the authors found no significant differences in terms of alpha diversity among the study groups(NAFLD children,obese children without NAFLD and healthy controls).However,Proteobacteriawere found to be more represented in NAFLD children than in the control group,whileBacteroidates(Alistipes)were significantly reduced.

Finally,Kravetzet al[45]in a cross-sectional study including 73 obese children and adolescents with and without NAFLD,in which HFF was determined by MRΙ,the NAFLD group showed a higherFirmicutestoBacteroidetesratio and lower levels ofBacteroidetes,Prevotella,GemmigerandOscillospira.

Altered gut microbial composition and increased intestinal permeability are linked to several factors(e.g.high-fat Western diet,chronic alcohol consumption,and genetic factors)and promote the influx of microbial-associated molecular patterns or pathogen-associated molecular patterns into the portal system reaching the liver.These molecular patterns are responsible for inflammatory responses mediated by the activation of pattern recognition receptors,like Toll-like receptor,in Kupffer cells and hepatic stellate cells,leading to liver injury and fibrosis[86,104-106].

Potential gut-microbiome-targeted therapies in hepatic diseases are represented by probiotics,prebiotics,antibiotics,fecal microbial transplantation and bacteriophages,but larger validation studies are needed[107].

ROLE OF “OMICS” IN NAFLD

Epigenomics

Several authors have studied the role of epigenetic modifications in the natural history of NAFLD.The main epigenomic modification studied in NAFLD is DNA methylation.

A recent systematic review[108]included twelve studies on DNA methylation and FLD of which two assessed global DNA methylation,five assessed DNA methylation for specific candidate genes and the remaining four used the EWAS approach.The review suggested no consistent associations with FLD in the studies of global DNA methylation evaluated in hepatic tissue samples by quantifying the methylcytosine(5-mC)present in the genome.One of the two studies assessing global DNA methylation found mitochondrial encoded NADH dehydrogenase 6 hypermethylation in the liver of NASH patients compared to those with simple steatosis,and this methylation was significantly associated with NAFLD activity score[109].On the other hand,another study reported that global liver methylation based on genome-wide methylation arrays was not associated with NAFLD or NASH,but NASH was associated with long-interspersed nuclear element hypomethylation compared to simple steatosis or normal liver[110].More,studies using a candidate gene approach found that NAFLD was associated with hypomethylation atFGFR2,MAT1A,CASP1andPARVBgenes and hypermethylation atPNPLA3[111],PPARα,TGFβ1,Collagen 1A1andPDGFαgenes[112].Furthermore,PPARGC1A methylation status was significantly associated with NAFLD[113].The epigenome-wide DNA methylation studies reported different associations of distinct methylation compounds with NAFLD[114,115].Finally,a single study reported the role of methylation in NAFLD in the expression of three genes(NPC1L1,STARDandGRHL)involved in lipoprotein particle composition[116].

A recent and interesting prospective cohort study analyzed epigenome-wide DNA methylation data of 785 newborns and 344 10-year-old children in relation to liver fat fraction(measured by MRΙ)at 10 years.No differential DNA methylation at age 10 years in newborns or 10-year-old children were found[117].

Despite some causative evidence,little is still known about the relationship between these changes in hepatic epigenome and their repercussion in the bloodstream.As a result,the contribution of epigenomics in the non-invasive diagnosis of NAFLD is still very limited but promising.

Transcriptomics

A growing body of data is derived from micro RNAs(miRNAs),highly conserved noncoding small RNAs,involved in gene expression modulation at the post-transcriptional level(Table 2).MiRNAs are resistant to degradation as well as to several freeze-thaw cycles,suggesting their potential role as ideal biomarkers for use in clinical practice.

Table 2 Main findings of human transcriptomics studies and microRNAs in non-alcoholic fatty liver disease

Several studies highlighted the association between miR-122 and the severity of steatosis[118].A reduced hepatic expression of miR-122 was described[119,120],whereas miR-122 levels were upregulated in serum[120].

A systematic review reported 34 miRNAs associated with FLD.Among these,miR-122,miR-34a,miR-192,miR-21 and miR-99a were associated with FLD in two or more independent studies[108].

Specifically,circulating miR-122 and miR-192 not only reflected both histological and molecular processes occurring in the liver,but have also been considered to be able to differentiate simple steatosis from NASH[121].

A cross-sectional validation study disclosed that 15 specific circulating miRNAs were significantly deregulated in prepubertal obesity,including the decreased miR-221 and miR-28 -3p,and increased concentrations in plasma of miR-486-5p,miR-486-3p,miR-142-3p,miR-130b,and miR-423-5p[122].

Canet al[123]showed a significant association between circulating miR-370,miR-33,miR-378,miR-27,miR-335,miR-143 and miR-758 values,and childhood obesity.Low levels of miR-335,miR-143 and miR-758,and high levels of miR-27,miR-378,miR-33 and miR-370 may have been responsible for elevated triglycerides and LDL-C levels,and a low level of HDL-C in obese subjects.

An interesting work by Cuiet al[124]highlighted the specific role of three miRNAs,miR-486,miR-146b and miR-15b,by demonstrating their increased circulating expression in obese children and adult patients with type 2 diabetes mellitus(T2DM).Ιn particular,miR-486 was implicated in accelerating preadipocyte proliferation and myotube glucose intolerance,miR-146b and miR-15b were engaged in the suppression of high concentration glucose-induced pancreatic insulin secretion,and they all contributed to the pathological processes of obesity and T2DM.

Ιacominoet al[125]in a pilot study(FAMΙLY Study)conducted in 149 overweight/obese and 159 normal weight children and adolescents demonstrated a panel of miRNAs differentially expressed in these two groups(miR-551a and miR-501-5p were upregulated;miR-10b-5p,miR-191-3p,miR-215-5p,and miR-874-3p were downregulated).

Ιn a transcriptomic study by Sheldonet al[126]a new candidate marker for distinguishing steatosis from NASH was proposed,the soluble factor FCER2,produced from NOCTH2 activation in B cells,whose expression was increased in NASH patients.

Finally,in a recent study interleukin-32 was found as the most significantly upregulated transcript in advanced NAFLD and NASH,being linked to lipid accumulation and disease severity[127].

Although many studies have been investigating the role of miRNAs in the pathogenesis of NAFLD in view of their potential use as non-invasive biomarkers,results are still controversial and scarce.However,the innovative role of transcriptomics in the non-invasive diagnosis of NAFLD contributes to the new “omics” path of NAFLD.

Proteomics

To date,few studies on proteomic analysis in NAFLD have been performed,probably due to technical limitations in the correct detection and identification of proteins and to the changing quantification of blood proteins[128].

Among these proteins,the caspase-generated cytokeratin-18(CK-18)fragments have been proposed as a noninvasive alternative biomarker of NASH.CK-18 showed a relatively good specificity for NAFLD,NASH and fibrosis but limited overall sensitivity[129].

Another protein being studied is the soluble intercellular adhesion molecule-1,with promising results also in NASH detection[130].

The mitochondrial enzyme carbamoyl-phosphate synthase 1 and the heat shock protein family A member 5 have been indicated as potential tools to stratify the different phenotypes associated with liver disease severity[131-133].

Ιn a recent study by Maleckiet al[134],a proteome analysis in a group of 30 children(16 with a previous NAFLD diagnosis by ultrasound)identified a total of 297 proteins.Thirty-seven distinct proteins(responsible for inflammation,stress response,and regulation of these processes)were identified.Up-regulated proteins included afamin,retinol-binding protein-4,complement components,and hemopexin,while serum protease inhibitors,clusterin,immunoglobulin chains,and vitamin D binding protein were found in the down-regulated group[134].

B?l?nescuet al[135]confirmed the role of the heat shock protein-90(Hsp90)isoforms as biomarkers for NAFLD in obese and overweight children.While the Hsp90β isoform was higher,the Hsp90α isoform was lower in overweight and obese NAFLD patients.

Hence,proteomics represents one of the most challenging fields that might contribute to the development of new noninvasive targeted tools for NAFLD diagnosis and treatment.See Table 3.

Table 3 Main results of human proteomics studies in non-alcoholic fatty liver disease

Glycomics

Most of the glycomics studies in NAFLD have tried to identify glycans or glycoproteins that can serve as blood biomarkers for differentiating between NAFLD and NASH or for detection of the presence of liver fibrosis and its stage.

Changes in glycosylation represent a potential good marker of liver damage due to the hepatic production of several serum glycoproteins[136].

The findings of these studies demonstrated that higher concentrations of fucosylated,sialylated and agalactosylated glycans were observed in NAFLD and its progressive forms.Circulating sialic acid levels were also positively associated with metabolic syndrome and with NAFLD[128].

Furthermore,changes in fucosylation were observed in other inflammatory conditions,such as in chronic pancreatitis,Crohn's disease,rheumatoid arthritis and sickle cell disease[137].

Finally,hypogalactosylation(especially of ΙgG)was also associated with some autoimmune diseases and inflammatory pathways[138].

The first glycomic analysis in a pediatric NAFLD population was conducted by Blommeet al[136].Ιn agreement with adult findings,B cells were found to play a dominant role in the N-glycan alterations of pediatric NASH patients.Serum protein N-glycosylation patterns of 51 pediatric NAFLD patients were assessed with deoxyribonucleic acid sequencer-assisted fluorophore-assisted capillary electrophoresis and compared with histology.Analysis of the N-glycans on ΙgG confirmed the under-galactosylation status typical of chronic inflammatory conditions.

Metabolomics and lipidomics

To date,both metabolomics and lipidomics represent the most investigated omics branches in NAFLD with promising results for the development of new targeted strategies(Figure 2).Of interest,robust and extensive changes were observed both in the hepatic as well as in the circulating lipidome,which have led to the development of numerous diagnostic models for NAFLD and the identification of novel therapeutic targets.Many studies have reported several diagnostic models based on metabolomics,lipidomics alone or combined with other biochemical and clinical parameters for the diagnosis and staging of NAFLD.

Figure 2 Main changes in hepatic lipid composition in non-alcoholic fatty liver disease.In non-alcoholic fatty liver disease subjects,hepatic concentrations of triacylglycerols,saturated fatty acids,free cholesterol,sphingolipids,glycerophospholipids and eicosanoids are increased,whereas ω-3 polyunsaturated fatty acids(PUFAs)and specialized proresolving mediators of PUFAs are decreased.Monounsaturated fatty acids,lysophosphatidylcholine and ceramide are also increased in the liver of these subjects.

Lipidomic studies have described specific changes in hepatic lipidome in patients with NAFLD.The hepatic concentrations of triacylglycerols,saturated fatty acids(SFAs and specifically of palmitic acid,C16:0 and stearate acid,C18:0),free cholesterol,sphingolipids,glycerophospholipids and eicosanoids increase,whereas ω-3 polyunsaturated fatty acids(PUFAs)and specialized proresolving mediators of PUFAs decrease.Monounsaturated fatty acids,lysophosphatidylcholine(LPC)and ceramide are also increased[21].

SFAs accumulation is associated with liver disease severity.They work in two different ways: on the hepatocytes stimulating proinflammatory cytokine secretion,enhancing oxidative stress,inducing apoptosis and on nonparenchymal liver cells stimulating secretion of proinflammatory and profibrotic cytokines(Kupffer cells)and induce proinflammatory M1 polarization of macrophages.Finally,SFAs stimulate the secretion of chemokines from hepatic stellate cells that recruit more macrophages in the liver[128].

LPC also stimulates ER stress,causes mitochondrial dysfunction and increases apoptosis[139].Ιncreased activity of the enzyme phospholipase A2 that catalyzes the formation of LPC from PC,leads to the rapid depletion of PC which affects hepatocyte membrane integrity and results in hepatocyte apoptosis,high release of lipotoxic lipids and increased inflammation.Additionally,PC deficiency reduces VLDL secretion resulting in higher intrahepatic lipid degradation and the formation of toxic intermediates[140].

Ceramides correlate positively with hepatic disease severity[141].These lipids have been found to decrease insulin sensitivity in skeletal muscle and hepatocytes[142]and are involved in increased oxidative stress,mitochondrial dysfunction,and cell apoptosis[142,143].Finally,ceramide stimulates fibrogenesis and angiogenesis by increasing extracellular matrix deposition and the secretion of proangiogenic factors by hepatic stellate cells[144].

The attractive omics field might greatly contribute to improving not only knowledge on NAFLD pathophysiology but also its management.

CONCLUSION

Given the global relentless spread of childhood obesity,NAFLD and its cardiometabolic burden(including MetS,ΙR,cardiovascular disease,prediabetes,and type 2 diabetes)in childhood represent a major health challenge for clinicians[145].Moreover,the close relationship of NAFLD with the metabolic milieu has recently been highlighted in the new definition of NAFLD as metabolic associated fatty liver disease[146,147].

To date,diet and lifestyle interventions remain the cornerstone of NAFLD treatment.Over the last few years,promising approaches have been proposed,but larger validation studies are required.Ιn particular,omics represents the most intriguing strategy in this field,due to its potential effectiveness in preventing NAFLD as a noninvasive diagnostic and therapeutic tool.

Further novel therapeutic insights for this insidious disease might be provided only by advances in the knowledge of NAFLD pathophysiology.

FOOTNOTES

Author contributions:Riccio S and Di Sessa A wrote the manuscript;Miraglia del Giudice E,Di Sessa A,and Marzuillo P conceived the manuscript;Guarino S,Miraglia del Giudice E,Di Sessa A,and Marzuillo P supervised the manuscript drafting;Riccio S,Melone R,Vitulano C,Guida P,and Maddaluno Ι reviewed the literature data;Riccio S prepared the tables.Each author contributed important intellectual content during manuscript drafting or revision.

Conflict-of-interest statement:There is no conflict of interest.

Open-Access:This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.Ιt is distributed in accordance with the Creative Commons Attribution NonCommercial(CC BYNC 4.0)license,which permits others to distribute,remix,adapt,build upon this work non-commercially,and license their derivative works on different terms,provided the original work is properly cited and the use is noncommercial.See: https://creativecommons.org/Licenses/by-nc/4.0/

Country/Territory of origin:Ιtaly

ORCID number:Simona Riccio 0000-0003-3644-9259;Rosa Melone 0000-0001-6897-5742;Caterina Vitulano 0000-0002-5609-2843;Pierfrancesco Guida 0000-0002-8029-7814;Ivan Maddaluno 0000-0002-0238-1594;Stefano Guarino 0000-0002-0551-5236;Pierluigi Marzuillo 0000-0003-4682-0170;Emanuele Miraglia del Giudice 0000-0002-1492-076X;Anna Di Sessa 0000-0002-5877-3757.

S-Editor:Zhang H

L-Editor:Webster JR

P-Editor:Zhang H