Biomarkers in Fatty Liver Disease—Here is the Skinny

Abstract

The study by Malecki et al.1Małecki et al.Adipocytokines as Risk Factors for Development of Nonalcoholic Fatty Liver Disease in Children.J CLin Exp Hepatol. 2021; https://doi.org/10.1016/j.jceh.2021.03.002Abstract Full Text Full Text PDF Scopus (1) Google Scholar represents a thoughtful undertaking regarding the use of adipokines as biomarkers in the evaluation of obesity-related fatty liver disease (NAFLD). They prospectively followed 73 children over two years, dividing patients into research and control groups based on ultrasound findings of the liver, wherein an abnormal study would result in further pursuit of biochemical analyses. Statistically significant differences in certain biomarkers were identified, particularly in that of adiponectin: lower levels of which were found in the NAFLD group and in children with elevated body mass indices (BMIs). The authors therefore concluded that adiponectin may be useful in evaluating risk of NAFLD and obesity in children. Adiponectin is secreted by adipose tissue in considerable quantities, modified from a 30-kDa monomeric protein into low-, middle-, and high-molecular-weight multimers, which this study did not differentiate. Once secreted into circulation, it binds to its receptor(s), prompting a cascade of reactions that influence insulin resistance.2Fang H. Judd R.L. Adiponectin regulation and function.Comp Physiol. 2018 Jun 18; 8 (PMID: 29978896): 1031-1063https://doi.org/10.1002/cphy.c170046Crossref PubMed Scopus (179) Google Scholar The concept of adipocytokines as biomarkers, particularly with that of adiponectin, has been extensively studied and debated. While some studies highlight adiponectin's anti-inflammatory properties and negative correlation with adiposity, there is also contrarian literature to support elevated adiponectin levels in chronic inflammatory and autoimmune diseases, unrelated to an increase in adipose tissue. Thus, there is the discerning thought that adiponectins may exhibit different functions depending on the individual metabolic milieu or specific metabolic syndrome–related comorbid condition.3Fantuzzi G. Adiponectin and inflammation: consensus and controversy.J Allergy Clin Immunol. 2008 Feb; 121 (Epub 2007 Dec 3. PMID: 18061654): 326-330https://doi.org/10.1016/j.jaci.2007.10.018Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar One limitation in this study was the use of ultrasound to diagnose NAFLD. Prior work has clearly established the low utility of ultrasound in the diagnosis of NAFLD. Most algorithms use ultrasound only to evaluate for anatomical abnormalities that may explain an abnormal biochemical liver profile, rather than to evaluate for fatty liver itself.4Mitsinikos Tania Kohli Rohit Pediatric nonalcoholic fatty liver disease.Clin Liver Disease. 2018; 11: 95-97https://doi.org/10.1002/cld.712Crossref Scopus (2) Google Scholar The use of ultrasound to divide patients into study versus control groups may have unintentionally excluded a large number of children with NAFLD missed by this intake modality. Liver biopsy has been established and continues to be the gold standard for diagnosing NAFLD,5Vos M.B. Abrams S.H. Barlow S.E. et al.NASPGHAN clinical practice guideline for the diagnosis and treatment of nonalcoholic fatty liver disease in children: recommendations from the expert committee on NAFLD (ECON) and the north American society of pediatric gastroenterology, hepatology and nutrition (NASPGHAN).J Pediatr Gastroenterol Nutr. 2017; 64: 319-334Crossref PubMed Scopus (310) Google Scholar yet only three patients in the study group underwent this procedure, which limits the broad application of the analyzed data and conclusions derived by the authors. A poignant question we would like to ask is whether adiposity necessarily reflects NAFLD? Although NAFLD was previously thought to occur primarily in obese individuals, it is now understood that there is a significant subgroup of individuals referred to as nonobese NAFLD or lean NAFLD. There is a growing body of evidence for alternative factors, including genetic predisposition, dietary and environmental influences, playing a key role in the pathogenesis of NAFLD. Patients with lean NAFLD often remain undiagnosed and asymptomatic. These patients typically have lower body weight, waist circumference, fasting glucose, and glycated hemoglobin than patients with obese NAFLD.6Wang A.Y. Dhaliwal J. Mouzaki M. Lean non-alcoholic fatty liver disease.Clin Nutr. 2019 Jun; 38 (Epub 2018 Aug 17. PMID: 30466956): 975-981https://doi.org/10.1016/j.clnu.2018.08.008Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar This is especially true in populations wherein low-BMI diabetes is prevalent, such as in China and India.7Das K. Das K. Mukherjee P.S. et al.Nonobese population in a developing country has a high prevalence of nonalcoholic fatty liver and significant liver disease.Hepatology. 2010; 51: 1593-1602Crossref PubMed Scopus (284) Google Scholar,8Eslam M. Chen F. George J. NAFLD in lean asians.Clin Liver Disease. 2020; 16: 240-243https://doi.org/10.1002/cld.930Crossref Scopus (1) Google Scholar Some studies compared patients with lean versus obese NAFLD and found that adiponectin levels were similar between both groups, and within normal range, findings that further lead us to question the potential for adiponectin in predicting fatty liver disease.9Kumar R. Prakash S. Chhabra S. et al.Association of pro-inflammatory cytokines, adipokines & oxidative stress with insulin resistance & non-alcoholic fatty liver disease.Indian J Med Res. 2012 Aug; 136: 229-236PubMed Google Scholar,10Yasutake K. Nakamuta M. Shima Y. et al.Nutritional investigation of non-obese patients with non-alcoholic fatty liver disease: the significance of dietary cholesterol.Scand J Gastroenterol. 2009; 44: 471-477Crossref PubMed Scopus (144) Google Scholar Furthermore, we agree with the authors that lower adiponectin levels may be more a “consequence of obesity,” suggesting that adiponectin may not necessarily correlate with NAFLD per se. This raises the critical question: does correlation imply causation? Although the pathogenesis of NAFLD has not been precisely established, there is now a better understanding of the complex molecular, genetic, and metabolic factors that contribute to development of disease. NAFLD pathogenesis can be summarized by the “multiple hit” hypothesis, which describes nutrition, insulin resistance, hormones, and gut microbiota as factors that act together on genetically predisposed persons to give rise to obesityrelated fatty liver disease.11Elena B. Massimo Emmanuel A.T. The multiple-hit pathogenesis OF non-alcoholic fatty liver DISEASE (NAFLD).Metabolism. 2016; 65: 1038-1048https://doi.org/10.1016/j.metabol.2015.12.012Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar Of these multiple “hits,” genetic predisposition plays a key role in the development of nonalcoholic steatohepatitis (NASH). Correlations in serum alanine aminotransferase (ALT), used as a marker of hepatic inflammation, among monozygotic twins were found to be higher than those of dizygotic twins.12Makkonen J. Pietilainen K.H. Rissanen A. Kaprio J. Yki-Jarvinen H. Genetic factors contribute to variation in serum alanine aminotransferase activity independent of obesity and alcohol: a study in monozygotic and dizygotic twins.J Hepatol. 2009; 50: 1035-1042Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar In addition, a cross-sectional analysis demonstrated a higher risk of NAFLD progression in first-degree relatives with hepatic fibrosis related to NAFLD.13Caussy C. Soni M. Cui J. et al.Nonalcoholic fatty liver disease with cirrhosis increases familial risk for advanced fibrosis.J Clin Invest. 2017; 127: 2697-2704Crossref PubMed Scopus (73) Google Scholar Cohort studies analyzing ethnicity demonstrated increased risk in the Hispanic population and lower risk in African Americans when corrected for all other modifiers including BMI. Furthermore, genome-wide association studies identified the most significant inherited risk factor for fatty liver disease: rs738409 C>G single-nucleotide polymorphism, which leads to the I148M protein variant of patatin-like phospholipase domain–containing 3 (PNPLA3). Recognized as the leading genetic determinant of fat content in the liver, the I148M protein variant was found more commonly in the Hispanic population.14Stefano R. Julia K. Chao X. et al.Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (1939) Google Scholar Genetics is also relevant to the impact of environmental factors, such as a subgroup showing significant response to a low-fructose diet.15Valerio N. Daniela L. Giorgio B. et al.Influence of dietary pattern, physical activity, and I148M PNPLA3 on steatosis severity in at-risk adolescents.Genes Nutr. 2014; 9: 392Crossref PubMed Scopus (42) Google Scholar In general, a high dietary intake of fructose, in the form of high-fructose corn syrup, has steadily increased over the years, increasing the risk of steatosis and fatty liver disease.16Vos M.B. Lavine J.E. Dietary fructose in nonalcoholic fatty liver disease.Hepatology. 2013; 57: 2525-2531https://doi.org/10.1002/hep.26299Crossref PubMed Scopus (207) Google Scholar The complex interplay between genetics and environment prompts the discussion of nature versus nurture in the development of obesity-related fatty liver disease. NAFLD describes a disease spectrum: simple steatosis (defined as a minimum of five percent hepatic steatosis), steatohepatitis, fibrosis, and cirrhosis. Simple steatosis follows a more benign course, whereas steatohepatitis associated with fibrosis can potentially progress to end-stage liver disease. Xanthakos et al. found that 18% of children with hepatosteatosis progress to steatohepatitis, whereas 11% progress to steatosis and fibrosis.17Xanthakos S.A. Lavine J.E. Yates K.P. et al.For the NASH clinical research network, progression of fatty liver disease in children receiving standard of care lifestyle advice.Gastroenterology. 2020 Nov; 159 (e10. https://doi.org/10.1053/j.gastro.2020.07.034): 1731-1751Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Therefore, although there is potential in those with steatosis to advance to fibrosis, disease does not always progress. Those who develop fibrosis may represent an extreme phenotype of disease, especially in children. Perhaps searching for alternative biomarkers that are sourced from hepatic physiology may be more relevant to NAFLD pathology than the focus on adipocytokines (Figure). A significant contributor to fatty liver disease is lipotoxic metabolite-induced oxidative stress, which leads to hepatocyte damage and death, inflammation, cytokine production, and fibrosis. Thus, hepatocyte damage is an important factor that helps drive the progression of NAFLD. The pathways involved in the NAFLD inflammatory process include JNK-AP-1, a protein kinase associated with apoptosis and steatohepatitis, as well as IKK-NF-kBD, a transcription factor that modulates inflammation.18Hotamisligil G.S. Inflammation and metabolic disorders.Nature. 2006; 444 ([PMID: 17167474): 860-867https://doi.org/10.1038/nature05485Crossref PubMed Scopus (5708) Google Scholar Subudhi et al. evaluated whole liver tissues in adults at high risk of fatty liver disease and identified patterns of gene expression and aberrant regulation in different stages of NAFLD. The study found notable changes in complement proteins in the progression of steatosis to steatohepatitis. Similarly, steatohepatitis was associated with increased gene expression of chemokine ligand 9 (CXCL9) and gamma-interferon-inducible lysosomal thiol reductase (GILT), the latter encoded by interferon-gamma-inducible protein 30. Only in the setting of fibrosis, genetic upregulation of extracellular matrix degrading proteinases and profibrotic genes, such as transforming growth factor beta 1 (TGFB1), was observed.19Subudhi S. Drescher H.K. Dichtel L.E. et al.Distinct hepatic gene-expression patterns of NAFLD in patients with obesity.Hepatol Commun. 2021; https://doi.org/10.1002/hep4.1789Crossref PubMed Scopus (1) Google Scholar Prior studies also report TGFB as a marker in fibrosis, playing an integral role in inflammation and NAFLD progression.20Nair Bhagyalakshmi Nath Lekshmi R. Inevitable role of TGF-β1 in progression of nonalcoholic fatty liver disease.J Recep Sign Transduc. 2020; https://doi.org/10.1080/10799893.2020.1726952Crossref PubMed Scopus (10) Google Scholar The other potential biomarkers for NAFLD include the interleukin-17 (IL-17) family of proinflammatory cytokines (IL-17 A-F): in both humans and mice, obesity was associated with increased IL-17A expression, suggesting that its accumulation in the liver may act on hepatic cells, augmenting proinflammatory cytokines that lead to production of reactive oxygen.21Giles D.A. Moreno-Fernandez M.E. Divanovic S. IL-17 Axis driven inflammation in non-alcoholic fatty liver disease progression.Curr Drug Targets. 2015; 16: 1315-1323https://doi.org/10.2174/1389450116666150531153627Crossref PubMed Scopus (43) Google Scholar Another factor of interest is fibroblast growth factor 19 (FGF19). The aberrant production or reduced hepatic response to FGF19 has been postulated to contribute to lipid metabolism dysfunction22Tim C.M.A.S. Hendrik A.M. Martin L. et al.The hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease and insulin resistance.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G440-G445Crossref PubMed Scopus (110) Google Scholar; furthermore, decreased levels of FGF19 were found to be associated with the development of fatty liver disease in obese adolescents, suggesting that FGF19 levels may also serve as a predictive risk factor for NAFLD.23Wojcik M. Janus D. Dolezal-Oltarzewska K. et al.A decrease in fasting FGF19 levels is associated with the development of non-alcoholic fatty liver disease in obese adolescents.J Pediatr Endocrinol Metab. 2012; 25 (PMID: 23329754): 1089-1093https://doi.org/10.1515/jpem-2012-0253Crossref PubMed Scopus (56) Google Scholar These and other similar specific inflammatory mediators and cytokines may serve as biomarkers to identify and monitor obesity-related fatty liver disease. In regard to biochemical biomarkers, Newton et al. monitored liver biopsies in children with NAFLD and found that serum ALT and gamma-glutamyl transferase (GGT) were associated with changes in histology and therefore may be better predictive indicators of histopathologic response.24Kimberly P.N. Joel E.L. Laura W. et al.Alanine aminotransferase and gamma-glutamyl transpeptidase predict histologic improvement in pediatric nonalcoholic steatohepatitis.Hepatology. 2020; 73: 937-951https://doi.org/10.1002/hep.31317Crossref PubMed Scopus (6) Google Scholar Studies on metabolomics have identified potential biomarkers of steatohepatitis in pediatric populations, including alpha-ketoglutarate, cysteine-glutathione disulfide, and N-acetylmethionine. Particularly, the upregulation of alpha-ketoglutarate, a derivative of glutamate, is among the most significant predictors of NASH, mirroring oxidative stress associated with the progression to steatohepatitis.25Kordy K. Li F. Lee D.J. et al.Metabolomic predictors of non-alcoholic steatohepatitis and advanced fibrosis in children.Front Microbiol. 2021; 12: 713234https://doi.org/10.3389/fmicb.2021.713234Crossref PubMed Scopus (1) Google Scholar In addition, there is a growing body of literature characterizing the role of altered bile acid synthesis in the pathogenesis of NAFLD. A prospective, cross-sectional study of adults with biopsy-confirmed fatty liver disease found a clinically significant rise in total fecal bile acids and serum bile acid markers in patients with fatty liver disease compared with healthy controls.26Mouzaki M. Wang A.Y. Bandsma R. et al.Bile acids and dysbiosis in non-alcoholic fatty liver disease.PLoS One. 2016; 11 (Published 2016 May 20)e0151829https://doi.org/10.1371/journal.pone.0151829Crossref PubMed Scopus (179) Google Scholar Although more data are needed to establish a correlation, particularly in children, the study of novel biomarkers such as bile acids or combinations of biomarkers (cellular, biochemical, and radiological) may be the way of the future (Figure). In conclusion, we surmise that adipocytokines, although tantalizing as an obesity-related cytokine, may, alas, not be the full answer, and perhaps not even the most optimal place to start, as a biomarker for NAFLD diagnosis, staging, and disease progression monitoring. We therefore advocate consideration of novel cytokines, biochemicals, and imaging modalities (Figure) to be defined as the ultimate biomarker(s) or holy grail of NAFLD-NASH. The authors have none to declare.