Valerio Nobili

Risk of severe liver disease in NAFLD with normal ALT levels: A pediatric report

We appreciated the interest in our article1 and the insightful comments from Dr. Enzo Emanuele. We believe that routine use of the proposed NASH scoring system, which comprises variables readily available to clinicians when they evaluate morbidly obese patients, can facilitate clinical decisions and help clinicians choose and tailor treatment for morbid obesity, including bariatric surgery. We agree with Dr. Emanuele that many other biomarkers may indeed increase the diagnostic performance of the proposed NASH scoring system. Further clinical translational research, including cost analysis, is needed to better define diagnostic and therapeutic approaches to obesity and its associated diseases.

Pathogen- or damage-associated molecular patterns during nonalcoholic fatty liver disease development.

S ensing danger is a key to an organism’s survival. The appropriate reaction is immediate and effective and consists of triggering molecular and cellular events that lead to elimination of the menace with tolerable collateral damage, through the process of inflammation. Invasive microbes cause an immediate inflammation with recruitment of innate immune cells to the site of pathogen entry, activation of phagocytosis, release of chemokines and cytokines, and initiation of the adaptive immune response. The same mechanisms are activated when tissue damage occurs because of trauma, ischemia/reperfusion injury, chemical toxicity, and cellular necrosis, all events that should be followed by wound-healing to ensure the maintenance of body and organ integrity. Also, in this case, cells of the innate immune system rush to the damaged area, releasing proinflammatory cytokines and chemokines to cause a phenomenon called sterile inflammation. If the trigger cannot be removed and the process becomes chronic, both infective and sterile inflammation cause substantial collateral damage. The continuous but ineffective actions of the innate immune system perpetuate tissue damage, with collagen deposition, fibrosis, and eventually permanent anatomical and functional alterations. The organism senses microbial infection through innate receptors encoded in the genome, called pattern-recognition receptors, including the Toll-like receptors (TLRs), the nucleotide-binding and oligomerization domain (NOD)-like receptors, and retinoic acid–inducible gene I (RIG-I)-like receptors. These receptors recognize pathogen-associated molecular patterns (PAMPs) expressed by bacteria, fungi, and viruses, but also bind damage-associated molecular patterns (DAMPs), which are molecules released by sterile injury. Thus, PAMPs and DAMPs that bind to the same type of receptors initiate identical intracellular pathways terminating in identical effector functions. Each member of the PAMP and DAMP families binds to one or more different TLRs (Table 1), and TLR expression varies among disparate cell types. Thus, the same PAMP or DAMP may have different effector functions depending on the cell type with which the interaction takes place. TLRs are type 1 transmembrane glycoproteins consisting of leucine-rich repeat motifs in the extracellular domain for ligand recognition, and a cytoplasmic Toll/ interleukin-1 (IL-1) receptor (TIR) intracellular domain indispensable for the activation of downstream signaling molecules. All TLRs ultimately activate the transcription factors nuclear factor kappa B (NF-jB) and activator protein-1 (AP-1) and thus the transcription of type 1 interferons and inflammatory cytokines. The proximal events of TLR signaling are initiated by the interaction of the TIR domain with cytosolic adaptor molecules. Myeloid differentiation factor 88 (MyD88) is the adaptor used by most TLRs, indispensable for the recruitment of the IL-1–associated kinases (IRAK1 and IRAK4). MyD88 is sufficient for signal transduction from TLR5, TLR7, TLR8, TLR9, and TLR11, but needs the cooperation of another adaptor, TIRAP (TIR domain–containing adaptor protein), downstream of TLR1-2, TLR2-6, and TLR4. Among all TLRs, TLR4 is the most peculiar, because it needs an accessory molecule, the glycoprotein MD-2, in order to effectively bind its best-known ligand, the bacterial endotoxin (lipopolysaccharide [LPS]). In addition, two independent signaling pathways are initiated by TLR4. Besides the TIRAP/MyD88-initiated cascade, a parallel signal runs through the TRAM (TRIF-related adaptor molecule) and Trif (TIR-domain–containing adapter-inducing interferon-b) adaptors, leading to the, albeit delayed, production of type 1 interferons. Stimulation of TLR4 via PAMPs such as LPS has been recently shown to play a central role in the development and progression of nonalcoholic fatty liver disease (NAFLD). On the contrary, the potential effect of DAMPs in NAFLD is still largely unexplored. Abbreviations: DAMP, damage-associated molecular pattern; FFA, free fatty acid; HFD, high-fat diet; HMGB1, high mobility group box 1; HSCs, hepatic stellate cells; IL, interleukin; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; NAFLD, nonalcoholic fatty liver disease; NF-jB, nuclear factor jB; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor. Address reprint requests to: Anna Alisi, Ph.D., Research Laboratories, Unit of Liver Research at ‘‘Bambino Gesù’’ Children’s Hospital, IRCCS, Piazza Sant’Onofrio 4, 00165 Rome, Italy. E-mail: anna.alisi@opbg.net; fax: þ39 06 68 59 29 04. CopyrightVC 2011 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.24611 Potential conflict of interest: Nothing to report.

Unraveling the genetics of fatty liver in obese children: Additive effect of P446L GCKR and I148M PNPLA3 polymorphisms†‡

N onalcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in children and adolescents in industrialized countries, mainly as a result of the epidemics of obesity, which in almost 80% of cases leads to fatty liver. Familial, epidemiological, and twin studies suggest that inherited factors play a major role in determining the susceptibility to develop both fatty liver and nonalcoholic steatohepatitis (NASH), and due to the lower number of confounding factors (such as disease duration, body fat, lifestyle habits, comorbidities, and drugs) and the likely more important role played by genetic factors in early onset disease, this is especially true for obese children. The demonstration that genetic variants of the patatinlike phospholipase domain-containing protein 3 (PNPLA3), and in particular the common rs738409 C>G single-nucleotide polymorphism (SNP) encoding for the I148M variant, are associated with hepatic fat content and increased liver enzymes, but also increase the risk of NASH and fibrosis progression, represented a landmark in the field. Furthermore, PNPLA3 genotype influenced the histological severity of NASH and fibrosis in obese pediatric patients (i.e., those also predisposed to potentially progressive liver disease), and the association with fibrosis was stronger than in adults. Still, a large fraction of steatosis heritability remained unexplained, until a recent genome-wide association study (GWAS) conducted in a large population was able to identify a wider set of genetic variants influencing steatosis (12), including I148M PNPLA3 and a SNP in glucokinase regulatory protein (GCKR), involved in the regulation of the uptake of monosaccharides and lipogenesis, and previously shown to influence serum levels of triglycerides. In addition, two SNPs in the promoter of apolipoprotein C3 (APOC3) were shown to influence liver fat accumulation and insulin resistance in male Indians, but no data were specifically available in the pediatric population. In this issue of HEPATOLOGY, Santoro et al. evaluate the combined effect of PNPLA3 rs738409, GCKR rs1260326, and APOC3 rs2854116 SNPs on hepatic fat content, insulin resistance, and lipoprotein levels in 455 obese children and adolescents of different ethnicity, 142 of whom underwent magnetic resonance imaging to quantify hepatic fat content. In line with previous data of the same authors, the I148M PNPLA3 variant was associated with fatty liver, but not with insulin resistance and dyslipidemia, whereas the GCKR rs1260326 SNP was associated with hepatic fat accumulation, large very low-density lipoproteins, and triglyceride levels. Furthermore, there was a joint effect of PNPLA3 and GCKR SNPs explaining 15%-32% of hepatic fat content variability according to ethnicity. On the contrary, APOC3 genotype did not influence hepatic fat content, insulin resistance, or dyslipidemia, as already indicated by recent studies in adults, thereby definitively discarding this variant as a major risk factor for steatosis and NASH. Therefore, the likely additive effect of PNPLA3 and GCKR variants explained almost one-third of hepatic fat content variance in obese children, although due to the limited number of subjects analyzed for each ethnicity, data should be replicated and the model of interaction re-evaluated in confirmatory cohorts. The rs1260326 GCKR encodes for the P446L protein variant, influencing the ability of GCKR to inhibit glucokinase in response to fructose-6-phosphate, thereby resulting in a constant increase in hepatic glucokinase activity and glucose uptake by the liver. Unrestricted hepatic glycolysis associated with the minor 446L allele leads on one hand to lower glucose and insulin levels, but on the other hand to increased levels of malonylCoA, which in turn may favor hepatic fat accumulation by serving as a substrate for lipogenesis and by blocking fatty acid oxidation through the inhibition of carnitinepalmytoil transferase-1. Figure 1 shows a possible simplified working model that may explain the major roles of the P446L GCKR variant in fatty liver. Abbreviations: APOC3, apolipoprotein C3; GCKR, glucokinase regulatory protein; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PNPLA3, patatin-like phospholipase domain-containing protein 3; SNP, single-nucleotide polymorphism. Address reprint requests to: Valerio Nobili, M.D., Valerio Nobili, Liver Research Unit, ‘‘Bambino Gesù,’’ Children’s Hospital and Research Institute, Piazza San Onofrio 4, 00168-I, Rome, Italy. E-mail: nobili66@yahoo.it; fax: (39) 6-6859-2904. CopyrightVC 2012 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.25617 Potential conflict of interest: Nothing to report.

Diagnostic Power of Fibroscan in Predicting Liver Fibrosis in Nonalcoholic Fatty Liver Disease

1. Ji J, Yamashita T, Budhu A, Forgues M, Jia HL, Li C, et al. Identification of microRNA-181 by genome-wide screening as a critical player in EpCAM-positive hepatic cancer stem cells. HEPATOLOGY 2009;50:472-480. 2. Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, et al. EpCAMpositive hepatocellular carcinoma cells are tumor-initiating cells with stem/ progenitor cell features. Gastroenterology 2009;136:1012-1024. 3. Yamashita T, Budhu A, Forgues M, Wang XW. Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma. Cancer Res 2007;67:10831-10839.

Profiling microRNA expression: A snapshot of nonalcoholic steatohepatitis and a recording of its pathogenesis

Virologic and histologic features of chronic hepatitis B virus-infected asymptomatic patients with persistently normal ALT. Gastroenterology 2008;134:1376-1384. 7. Lai M, Hyatt BJ, Nasser I, Curry M, Afdhal NH. The clinical significance of persistently normal ALT in chronic hepatitis B infection. J Hepatol 2007;47:760-767. 8. Lin CL, Liao LY, Liu CJ, Yu MW, Chen PJ, Lai MY, et al. Hepatitis B viral factors in HBeAg-negative carriers with persistently normal serum alanine aminotransferase levels. HEPATOLOGY 2007;45:1193-1198. 9. Martinot-Peignoux M, Boyer N, Colombat M, Akremi R, Pham B-N, Ollivier S, et al. Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers. J Hepatol 2002;36:543-548. 10. Feld JJ, Ayers M, El-Ashry D, Mazzulli T, Tellier R, Heathcote EJ. Hepatitis B virus DNA prediction rules for hepatitis B e antigen-negative chronic hepatitis B. HEPATOLOGY 2007;46:1057-1070.

Portal inflammation as index of steatohepatitis in children with nonalcoholic fatty liver disease.

1. Zhao H, Miao R. A call for attention and trials on HBeAg-negative, ALTnormal chronic hepatitis B virus infection. HEPATOLOGY 2009; doi: 10.1002/hep.23110. 2. Degertekin B, Lok AS. Indications for therapy in hepatitis B. HEPATOLOGY 2009;49(5 Suppl.):S129-S137. 3. Pharmasset voluntarily halts clinical studies with clevudine in hepatitis B infected patients [press release]. Princeton, NJ: Pharmasset Inc.; April 20, 2009. http://investor.pharmasset.com/releasedetail.cfm?ReleaseID 378789. Accessed June 2009. 4. Seok J, Lee DK, Lee CH, Park MS, Kim SY, Kim HS, et al. Long-term therapy with clevudine for chronic hepatitis B can be associated with myopathy characterized by depletion of mitochondrial DNA. HEPATOLOGY 2009;49:2080-2086. 5. Kim BK, Oh J, Kwon SY, Choe WH, Ko SY, Rhee KH, et al. Clevudine myopathy in patients with chronic hepatitis B. J Hepatol 2009; doi: 10.1016/j.jhep.2009.04.019.

Hedgehog/hyaluronic acid interaction network in nonalcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma

We read with great interest the article published in HEPATOLOGY by Guy and colleagues. In this study, the authors demonstrated a direct correlation between the hepatic level of Hedgehog (HH) pathway activity and the severity of liver injury and fibrosis in human nonalcoholic fatty liver disease (NAFLD). The expression of Sonic Hedgehog (SHH), a ligand of the HH pathway, was analyzed by immunohistochemistry in 84 NAFLD patients with different stages of fibrosis. In these patients, SHH expression increases concomitantly to fibrosis stage, ballooning, portal inflammation, and fibrosis. Interestingly, at the univariate analysis age, body mass index, waist circumference, homeostasis model assessment-insulin resistance, essential hypertension, and fibrosis stage strongly correlated with hepatic expression of SHH. In HH signaling, the interaction of SHH with the cell surface receptor Patched depresses Smoothened (SMO) activity, leading to nuclear localization of glioblastoma family transcription factors (GLI1, 2, and 3) that regulate the expression of cell-specific target genes. Interestingly, Guy and colleagues observed a significant correlation between nuclear accumulation of GLI2, liver fibrosis, and other risk factors for NAFLD. Accordingly, we found that GLI2 was overexpressed in liver extracts from rats treated with high fat/high fructose (HF/HFr) diet as compared with standard diet (Fig. 1A). We previously demonstrated that rats fed a HF/HFr diet histologically resemble human NAFLD, developing a rare fibrosis with increased collagen VI. Here we show that this dietetic regimen also increases hyaluronic acid (HA) circulating levels (Fig. 1B). HA, as well as osteopontin, is an important ligand for CD44, a marker of cancer stem cells, whose expression is inhibited by SMO antagonists. We hypothesize that an interaction network may exist between HA and HH signaling. This hypothesis is strongly supported by data from Patched1 mutant mice (Ptch1þ/ ), in which the HH pathway is constitutively activated and displays high levels of circulating HA with respect to Ptch1þ/þ mice (Fig. 1c). These results may explain why these mice are susceptible to developing fibrosis in diet-induced NAFLD.

Utility of magnetic resonance imaging in the evaluation of hepatic fat content

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Metabolic syndrome and nonalcoholic steatohepatitis recurrence after liver transplantation in children

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in children in the Western world. NAFLD encompasses a broad spectrum of liver abnormalities, which range from simple fatty liver disease to nonalcoholic steatohepatitis (NASH), with various degrees of inflammation and fibrosis, that can progress to cryptogenic cirrhosis. Pediatric NAFLD is also frequently associated with central obesity hypertension, hyperlipidemia, insulin resistance, and type 2 diabetes mellitus. All these conditions are part of a well-defined cluster known as metabolic syndrome. Metabolic syndrome and NASH are connected by the fact that they are complex, multifactorial diseases characterized by a common network of pathogenetic mechanisms, which include genetic background factors (eg, gene polymorphisms) and prenatal and postnatal factors (eg, the intrauterine environment, diet, and lifestyle). Furthermore, some epidemiological studies support a close association between metabolic syndrome and NAFLD in both adults and children. NAFLD/NASH and related fibrosis are recognized as potential causes of end-stage liver disease. The indication to treat endstage liver disease due to NAFLD/NASH with orthotopic liver transplantation is increasing in clinical practice. In fact, fatty liver disease in conjunction with the obesity epidemic compromises posttransplant outcomes and increases the risk of NAFLD/ NASH recurrence. Notably, the discrepancy between the availability of organs for transplantation and the demand for them has prompted many centers to accept livers previously considered inadequate in terms of posttransplant survival and functionality. Some centers consider only grafts with less than 10% steatosis to be acceptable because the use of moderately steatotic grafts is believed to increase the risk of primary nonfunction of the liver after liver transplantation. Furthermore, the use of fatty liver donors increases the risk of de novo NAFLD/NASH. In an interesting study recently published in this journal, Laish et al. evaluated the prevalence of metabolic syndrome in 252 liver transplant recipients (mean age 1⁄4 54.5 6 2.8 years). The authors demonstrated that the percentages of patients with a body mass index > 30 kg/m, hypertriglyceridemia (>150 mg/dL), low levels of high-density lipoprotein cholesterol (<40 mg/dL for men and <50 mg/dL for women), hypertension, and diabetes were significantly higher after liver transplantation with respect to the baseline values. The authors concluded that because the frequency of metabolic syndrome in liver transplant recipients is twice that in the general population, further prospective studies are required to determine appropriate clinical management. Interestingly, another recent cohort study of 88 individuals undergoing liver transplantation for NAFLDrelated cirrhosis has demonstrated that NAFLD recurrence is common in the first 5 years after transplantation and is closely associated with features of metabolic syndrome, such as high body mass indices and increased triglyceride levels. There are no long-term follow-up or prospective studies of metabolic syndrome and NAFLD/NASH in pediatric liver transplant recipients, although several factors, such as gene polymorphisms, nutritional status, and long-term exposure to drug toxicity (eg, immunosuppressive therapy), may contribute to metabolic syndrome and NAFLD in this group. On the basis of this evidence, we believe that not only pretransplant strategies for monitoring the metabolism and histology of donor livers but also personalized posttransplant therapeutic approaches for preventing metabolic syndrome and NAFLD/NASH should be considered for children. As we recently reported, we strongly believe that further studying the association between NAFLD and metabolic syndrome in liver graft donors and pediatric transplant recipients is necessary; likewise, we should examine the complex relationship between the genes of donors and recipients, the metabolism of recipients, and the

Reduced cardio-respiratory fitness in obesity with and without nonalcoholic fatty liver disease.

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