PERSPECTIVE
METABOLIC SYNDROME AND RELATED DISORDERS Volume X, Number X, 2015 Mary Ann Liebert, Inc. Pp. 1–4 DOI: 10.1089/met.2015.1502
A Perspective on Metabolic Syndrome and Nonalcoholic Fatty Liver Disease Giovanni Targher, MD,1 and Christopher D. Byrne, MBBCh 2,3
Abstract
Nonalcoholic fatty liver disease (NAFLD) is increasingly being diagnosed worldwide and is strongly associated with the features of metabolic syndrome. In this brief review, we discuss two key questions relating to NAFLD and metabolic syndrome: (1) Does NAFLD predict the development of type 2 diabetes mellitus and metabolic syndrome, or is it simply an epiphenomenon? (2) Are there differences between metabolic syndrome–associated NAFLD and NAFLD associated with genetic variation in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene? Accumulating evidence indicates that NAFLD is not simply the hepatic manifestation of the metabolic syndrome, but is a pathogenic determinant of the syndrome. Genetic variation within the PNPLA3 gene (e.g., I148M homozygosity) confers a higher risk of developing more severe histological features of NAFLD, but a lower risk of developing metabolic syndrome traits. We suggest that future research is now required to elucidate whether both metabolic syndrome–related NAFLD and PNPLA3-related NAFLD produce the same risk of developing extrahepatic complications. the assertion that the conventional paradigm of NAFLD representing the mere ‘‘hepatic manifestation’’ of the metabolic syndrome is outdated, and it is now becoming clear that NAFLD is also a pathogenic determinant of metabolic syndrome.1 With regard to this evolving concept, there is a growing body of evidence strongly supporting the notion that NAFLD precedes the development of T2DM and metabolic syndrome.4 There are now approximately 30 longitudinal, retrospective, and prospective studies that provide strong evidence for NAFLD as an independent predictor of the future development of T2DM.4–15 Moreover, more than a dozen of longitudinal studies with a relatively long duration of follow-up reported that NAFLD, as diagnosed by either ultrasonography or biochemistry, is independently associated with an increased incidence of several components of metabolic syndrome, not only of T2DM.4,16–24 Amongst the prospective studies that have used ultrasonography to diagnose NAFLD and that have assessed the risk of developing T2DM, the risk of T2DM varied markedly from an approximately 60% increase to approximately a sixfold increase in risk.7–15 This wide interstudy variation in T2DM risk might reflect differences in NAFLD severity, as the study by Park et al.13 showed that the incidence rate of
Introduction
N
onalcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease of this decade, which is in part correlated with the global epidemic of obesity, the metabolic syndrome, and type 2 diabetes mellitus (T2DM).1,2 Cardiovascular disease (CVD) is the leading cause of death in NAFLD,3 followed by malignancy and liver failure, all of which are predominantly due to the progressive form of NAFLD, namely nonalcoholic steatohepatitis.1 The last few years have seen advances in many aspects of NAFLD, including molecular mechanisms involved in hepatic lipotoxicity and insulin resistance (IR), mechanisms of CVD risk, identification of novel gene associations, noninvasive assessment, and therapeutic agents. Here, we highlight some of these findings that are likely to have a major influence on the field.
Does NAFLD Predict the Development of T2DM and Metabolic Syndrome? NAFLD is now increasingly diagnosed in individuals who do not have T2DM or metabolic syndrome. This supports
1 Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University and Azienda Ospedaliera Universitaria Integrata of Verona, Verona, Italy. 2 Nutrition and Metabolism, Faculty of Medicine, University of Southampton, Southampton, United Kingdom. 3 Southampton National Institute for Health Research Biomedical Research Centre, University Hospital Southampton, Southampton, United Kingdom.
1
2
T2DM increased sharply according to the ultrasonographic severity of NAFLD at baseline (normal, 7%; mild, 9.8%; moderate-to-severe, 17.8%). Even after adjustment for multiple confounders, the hazard ratios (HRs) for T2DM development were higher in the mild-NAFLD group [adjusted HR, 1.09; 95% confidence interval (CI) 0.8–1.5] and in the moderate-to-severe-NAFLD group (adjusted HR, 1.73; 95% CI 1.0–3.0) compared with the no-NAFLD group.13 Of note, Sung et al. have investigated the impact of abdominal obesity, IR, and hepatic steatosis on the risk of T2DM at the 5-year follow-up in a cohort of over 12,000 Korean individuals.14 These data showed that each of these three risk factors was associated with T2DM risk, and each risk factor was associated with an approximate doubling of the T2DM risk after adjustment for other established risk factors. Notably, in the same observational cohort of individuals, the authors examined the impact of resolution of hepatic steatosis on the risk of T2DM at the 5-year followup to establish whether NAFLD improvement was associated with T2DM risk reduction. These data demonstrated that there was a significant T2DM risk reduction in those subjects in whom hepatic steatosis on ultrasonography resolved over time. In particular, in those subjects, risk of T2DM decreased to the background risk of someone who had never had hepatic steatosis.15 Conversely, the individuals in whom the severity of hepatic steatosis worsened over 5 years showed a marked increase in T2DM risk [adjusted odds ratio (OR) = 6.13; 95% CI 2.5–14.6 compared with the risk in people with resolution of hepatic steatosis], further supporting the notion that more severe forms of NAFLD are associated with a greater risk of T2DM.15 From these published data it is important to understand how mechanistically pre-existent NAFLD may lead to the development of T2DM and metabolic syndrome. With the exception of cases where NAFLD results from either familial hypobetalipoproteinemia or patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms in which NAFLD is usually dissociated from IR,25,26 NAFLD directly causes hepatic IR in most cases. Molecular and genetic studies have recently supported the view that the pathogenetic mechanisms contributing to intrahepatocytic lipid compartmentation are determinants of whether fatty liver is or is not associated with IR and metabolic syndrome.27 Proposed mechanisms by which NAFLD causes hepatic IR implicate various lipid species, inflammatory signaling, and other cellular modifications.27 Experimental studies have elucidated a key role for hepatic diacylglycerol activation of protein kinase Ce (PKCe) in triggering hepatic IR.28 Alterations in the regulation of intrahepatic lipid droplet metabolism influence the intracellular compartmentation of diacylglycerol, which dictates whether or not PKCe will translocate to the plasma membrane so promoting lipotoxicity and hepatic IR.27,28
Are There Differences Between Metabolic Syndrome–Related NAFLD and PNPLA3-Related NAFLD? Although, as discussed above, NAFLD is strongly associated with metabolic syndrome features,1,2 there are some people who develop NAFLD but do not have metabolic syndrome.29 This suggests that other factors besides metabolic syndrome– specific mechanisms are involved in NAFLD pathogenesis.
TARGHER AND BYRNE
To date, the strongest evidence that any single gene may cause NAFLD, independently of metabolic syndrome, is with a specific polymorphism in the PNPLA3 gene. Increased hepatic fat content30,31 and, in contrast to metabolic syndrome, low circulating serum triglycerides (TG) levels and a dissociation between hepatic steatosis and IR26,32 have been described with the GG allele (I148M) gene variant of PNPLA3 in people with NAFLD. Although there is modest attenuation of metabolic syndrome features, such as improved IR and decreased TG levels, with the GG genotype, it is uncertain whether this may also affect risk of CVD with NAFLD. Perhaps surprisingly, it was recently reported that in patients with NAFLD there was a greater prevalence of increased carotid intima media thickness among those with the GG genotype compared to those with the combined CG + CC genotypes.33 However, further studies are needed to elucidate this issue and determine whether the NAFLD phenotype occurring with the GG allele gene variant is associated with higher or lower risk of CVD. The precise function of PNPLA3 is uncertain, but both hepatic lipogenic and lipolytic roles have been proposed.34 PNPLA3 has acyltransferase activity, converting lysophosphatidic acid to phosphatidic acid within the triacylglycerol synthesis pathway, and also has triacylglycerol hydrolysis activity. It has been suggested that homozygosity of a G / C substitution at position 148 by changing an isoleucine to a methionine increases lipogenic activity and decreases hydrolytic activity. However, elucidating the precise function of hepatic PNPLA3 in NAFLD and investigating the influence of I148M has proved difficult. Until recently, animal studies have also provided limited insight. Very recent work with the I148M PNPLA3 knockin mice has shown that, although these mice have normal liver fat levels on a chow diet, when fed a high-sucrose diet, their liver fat levels increased two- to three-fold.35 The increased liver fat content in these knockin mice was also accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 mRNA.35 These data provide the first direct evidence that physiological expression of the PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is very important and associated with the accumulation of lipid in the liver. The strength of effect of the I148M variant on NAFLD disease severity across different populations has been analyzed in a systematic review and meta-analysis of 16 studies.36 In this analysis, homozygosity for I148M was associated with *75% higher hepatic fat content compared with the CC variant. The I148M GG variant was also associated with more severe NAFLD. For example, GG homozygotes had higher serum aminotransferase levels and an approximately three-fold greater risk of hepatic necroinflammation and fibrosis when compared with CC homozygous individuals. Evaluation of the risk associated with heterozygosity for the I148M variant suggested that the additive genetic model best explained the effect of I148M on the susceptibility to develop NAFLD. Nevertheless, and surprisingly, carrying two G alleles did not seem to increase the risk of severe histological features. Interestingly, metaregression showed an inverse association between male sex and the effect of I148M on hepatic fat content,36 suggesting that there may be dissimilar influences of I148M in men and women.
METABOLIC SYNDROME AND NAFLD
Although decreased serum TG levels32 have been described with the GG variant, Qiao et al. showed in an animal model that PNPLA3 increased lipogenesis, inducing hepatic verylow-density lipoprotein (VLDL)–TG accumulation that, in turn, may be secreted into serum, thereby increasing serum TG levels.37 The mechanisms by which PNPLA3 regulates VLDL assembly and secretion are poorly understood. PNPLA3 might alter the availability of hepatic lipid for VLDL assembly and secretion, thereby affecting serum TG concentrations. Similarly by regulating hepatic lipolytic activity, PNPLA3 might alter the availability of hepatic TG for VLDL assembly and secretion, thereby also affecting serum TG levels. There is some evidence that the PNPLA3 I148M GG genotype facilitates incorporation into liver lipid droplets of different types of fatty acid (e.g., palmitoleic acid), thereby altering the fatty acid composition of the lipid droplets.38 Whether the PNPLA3 genotype influences the fluxes of hepatic fatty acids to affect liver fat and VLDL assembly and secretion in NAFLD is uncertain. Recently, Nobili et al. showed that the PNPLA3 GG genotype diminished the beneficial effect of the long-chain omega-3 polyunsaturated fatty acid docosahexanoic acid (DHA) treatment on liver disease end points in children with NAFLD.39 DHA and eicosapentaenoic acid (EPA) are known to exert hypotriglyceridemic effects,40 and we have recently also shown a benefit of high tissue enrichment with DHA with omega-3 fatty acid treatment to decrease hepatic fat content in NAFLD.41 The hypotriglyceridemic effects of omega-3 fatty acids have also been demonstrated in NAFLD patients,42 and it is known that omega-3 fatty acids reduce hepatic VLDL–TG secretion via apolipoprotein B100 degradation,43 downregulation of de novo TG synthesis,44 enhancement of liver b-oxidation, and reduction of adipose tissue inflammation, thus reducing plasma nonesterified fatty acid delivery from adipose tissue to the liver.45 We suggest further work is needed to understand how PNPLA3 I148M modifies VLDL assembly (and/or secretion) and how PNPLA3 I148M modifies fluxes of omega-3 fatty acids in particular to affect liver disease severity and to affect metabolic syndrome features, such as increased serum TG concentrations in NAFLD.
Conclusions The current evidence supports a key role of NAFLD in the pathogenesis of hepatic IR and T2DM. NAFLD may also have relevant clinical implications for diagnosing, preventing, and treating the metabolic syndrome. Additionally, we believe that considerable attention should be paid to the development of NAFLD in the absence of metabolic syndrome and in the absence of overweight/obesity (i.e., the so called ‘‘lean’’ NAFLD). A key unanswered question is whether ‘‘lean’’ NAFLD and PNPLA3-related NAFLD carry the same risk as NAFLD occurring with metabolic syndrome for the development of extrahepatic (CVD and noncardiovascular) complications. Future well-designed prospective studies will be needed to answer this question.
Acknowledgments G.T. is supported in part by grants from the University School of Medicine of Verona. C.D.B. is supported in part by the Southampton National Institute for Health Research Biomedical Research Centre.
3
Author Disclosure Statement No competing financial interests exist.
References 1. Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol 2013;10:330–344. 2. Targher G, Byrne CD. Clinical review: Nonalcoholic fatty liver disease: A novel cardiometabolic risk factor for type 2 diabetes and its complications. J Clin Endocrinol Metab 2013;98:483–495. 3. Targher G, Day CP, Bonora E. Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease. N Engl J Med 2010;363:1341–1350. 4. Lonardo A, Ballestri S, Marchesini G, et al. Nonalcoholic fatty liver disease: A precursor of the metabolic syndrome. Dig Liv Dis 2014; doi: http://dx.doi.org/10.1016/j.dld.2014 .09.020. 5. Fraser A, Harris R, Sattar N, et al. Alanine aminotransferase, gamma-glutamyltransferase, and incident diabetes: The British Women’s Heart and Health Study and metaanalysis. Diabetes Care 2009;32:741–750. 6. Balkau B, Lange C, Vol S, et al.; Group Study D.E.S.I.R. Nine-year incident diabetes is predicted by fatty liver indices: The French D.E.S.I.R. study. BMC Gastroenterol 2010;10:56. 7. Shibata M, Kihara Y, Taguchi M, et al. Nonalcoholic fatty liver disease is a risk factor for type 2 diabetes in middleaged Japanese men. Diabetes Care 2007;30:2940–2944. 8. Yamada T, Fukatsu M, Suzuki S, et al. Fatty liver predicts impaired fasting glucose and type 2 diabetes mellitus in Japanese undergoing a health checkup. J Gastroenterol Hepatol 2010;25:352–356. 9. Bae JC, Rhee EJ, Lee WY, et al. Combined effect of nonalcoholic fatty liver disease and impaired fasting glucose on the development of type 2 diabetes: A 4-year retrospective longitudinal study. Diabetes Care 2011;34:727–729. 10. Chon CW, Kim BS, Cho YK, et al. Effect of nonalcoholic fatty liver disease on the development of type 2 diabetes in nonobese, nondiabetic korean men. Gut Liver 2012;6:368–373. 11. Choi JH, Rhee EJ, Bae JC, et al. Increased risk of type 2 diabetes in subjects with both elevated liver enzymes and ultrasonographically diagnosed nonalcoholic fatty liver disease: A 4-year longitudinal study. Arch Med Res 2013;44:115–120. 12. Chang Y, Jung HS, Yun KE, et al. Cohort study of nonalcoholic fatty liver disease, NAFLD fibrosis score, and the risk of incident diabetes in a Korean population. Am J Gastroenterol 2013;108:1861–1868. 13. Park SK, Seo MH, Shin HC, et al. Clinical availability of nonalcoholic fatty liver disease as an early predictor of type 2 diabetes mellitus in Korean men: 5-Year prospective cohort study. Hepatology 2013;57:1378–1383. 14. Sung KC, Jeong WS, Wild SH, et al. Combined influence of insulin resistance, overweight/obesity, and fatty liver as risk factors for type 2 diabetes. Diabetes Care 2012;35: 717–722. 15. Sung KC, Wild SH, Byrne CD. Resolution of fatty liver and risk of incident diabetes. J Clin Endocrinol Metab 2013;98: 3637–3643. 16. Jo SK, Lee WY, Rhee EJ, et al. Serum gamma-glutamyl transferase activity predicts future development of metabolic syndrome defined by 2 different criteria. Clin Chim Acta 2009;403:234–240.
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17. Adams LA, Waters OR, Knuiman MW, et al. NAFLD as a risk factor for the development of diabetes and the metabolic syndrome: An eleven-year follow-up study. Am J Gastroenterol 2009;104:861–867. 18. Xu Y, Bi YF, Xu M, et al. Cross-sectional and longitudinal association of serum alanine aminotransaminase and g-glutamyltransferase with metabolic syndrome in middleaged and elderly Chinese people. J Diabetes 2011;3:38–47. 19. Liu CF, Zhou WN, Fang NY. Gamma-glutamyltransferase levels and risk of metabolic syndrome: A meta-analysis of prospective cohort studies. Int J Clin Pract 2012;66:692–698. 20. Ryoo JH, Choi JM, Moon SY, et al. The clinical availability of non alcoholic fatty liver disease as an early predictor of the metabolic syndrome in Korean men: 5-Year’s prospective cohort study. Atherosclerosis 2013;227:398–403. 21. Suh YJ, Park SK, Choi JM, et al. The clinical importance of serum g-glutamyltransferase level as an early predictor of obesity development in Korean men. Atherosclerosis 2013; 227:437–441. 22. Cicero AF, D’Addato S, Reggi A, et al.; Brisighella Heart Study. Gender difference in hepatic steatosis index and lipid accumulation product ability to predict incident metabolic syndrome in the historical cohort of the Brisighella Heart Study. Metab Syndr Relat Disord 2013;11:412–416. 23. Ryoo JH, Oh CM, Kim HS, et al. Clinical association between serum g-glutamyltransferase levels and the development of insulin resistance in Korean men: A 5-year follow-up study. Diabet Med 2014;31:455–461. 24. Sung KC, Wild SH, Byrne CD. Development of new fatty liver, or resolution of existing fatty liver, over five years of follow up, and risk of incident hypertension. J Hepatol 2014;60:1040–1045. 25. Amaro A, Fabbrini E, Kars M, et al. Dissociation between intrahepatic triglyceride content and insulin resistance in familial hypobetalipoproteinemia. Gastroenterology 2010;139:149– 153. 26. Kantartzis K, Peter A, Machicao F, et al. Dissociation between fatty liver and insulin resistance in humans carrying a variant of the patatin-like phospholipase 3 gene. Diabetes 2009;58:2616–2623. 27. Birkenfeld AL, Shulman GI. Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology 2014;59:713–723. 28. Perry RJ, Samuel VT, Petersen KF, et al. The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature 2014;510:84–91. 29. Sung KC, Cha SC, Sung JW, et al. Metabolically healthy obese subjects are at risk of fatty liver but not of preclinical atherosclerosis. Nutr Metab Cardiovasc Dis 2014;24:256–262. 30. Romeo S, Kozlitina J, Xing C, et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2008;40:1461–1465. 31. Mashek DG, Greenberg AS. Serum TAG analysis differentiates between genetic and obesity-associated NAFLD. Diabetes 2014;63:42–44. 32. Hyysalo J Gopalacharyulu P, Bian H, et al. Circulating triacylglycerol signatures in nonalcoholic fatty liver disease associated with the I148M variant in PNPLA3 and with obesity. Diabetes 2014;63:312–322.
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33. Petta S, Valenti L, Marchesini G, et al. PNPLA3 GG genotype and carotid atherosclerosis in patients with nonalcoholic fatty liver disease. PLoS One 2013;8:e74089. 34. Farrell GC. PNPLeAse get the fats right: Does lipogenesis or lipolysis cause NASH? Hepatology 2010;52:818–821. 35. Smagris E, BasuRay S, Li J, et al. Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis. Hepatology 2015;61:108–118. 36. Sookoian S, Pirola CJ. Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology 2011;53:1883–1894. 37. Qiao A, Liang J, Ke Y, et al. Mouse patatin-like phospholipase domain-containing 3 influences systemic lipid and glucose homeostasis. Hepatology 2011;54:509–521. 38. Ruhanen H, Perttila¨ J, Ho¨ltta¨-Vuori M, et al. PNPLA3 mediates hepatocyte triacylglycerol remodeling. J Lipid Res 2014;55:739–746. 39. Nobili V, Bedogni G, Donati B, et al. The I148M variant of PNPLA3 reduces the response to docosahexaenoic acid in children with non-alcoholic fatty liver disease. J Med Food 2013;16:957–960. 40. Shearer GC, Savinova OV, Harris WS. Fish oil—How does it reduce plasma triglycerides? Biochim Biophys Acta 2012;1821:843–851. 41. Scorletti E, Bhatia L, McCormick KG, et al.; on behalf of the WELCOME Study Investigators. Effects of purified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease: Results from the WELCOME study. Hepatology 2014;60:1211–1221. 42. Spadaro L, Magliocco O, Spampinato D, et al. Effects of n-3 polyunsaturated fatty acids in subjects with nonalcoholic fatty liver disease. Dig Liver Dis 2008;40:194– 199. 43. Maitin V, Andreo U, Guo L, et al. Docosahexaenoic acid impairs the maturation of very low density lipoproteins in rat hepatic cells. J Lipid Res 2014;55:75–84. 44. Tanaka N, Zhang X, Sugiyama E, et al. Eicosapentaenoic acid improves hepatic steatosis independent of PPAR-alpha activation through inhibition of SREBP-1 maturation in mice. Biochem Pharmacol 2010;80:1601–1612. 45. Scorletti E, Byrne CD. Omega-3 fatty acids, hepatic lipid metabolism, and nonalcoholic fatty liver disease. Annu Rev Nutr 2013;33:231–248.
Address correspondence to: Prof. Giovanni Targher, MD Division of Endocrinology, Diabetes and Metabolism Department of Medicine University and Azienda Ospedaliera Universitaria Integrata Piazzale Stefani, 1 37126 Verona Italy E-mail: [email protected]
METABOLIC SYNDROME AND RELATED DISORDERS Volume X, Number X, 2015 Mary Ann Liebert, Inc. Pp. 1–4 DOI: 10.1089/met.2015.1502
A Perspective on Metabolic Syndrome and Nonalcoholic Fatty Liver Disease Giovanni Targher, MD,1 and Christopher D. Byrne, MBBCh 2,3
Abstract
Nonalcoholic fatty liver disease (NAFLD) is increasingly being diagnosed worldwide and is strongly associated with the features of metabolic syndrome. In this brief review, we discuss two key questions relating to NAFLD and metabolic syndrome: (1) Does NAFLD predict the development of type 2 diabetes mellitus and metabolic syndrome, or is it simply an epiphenomenon? (2) Are there differences between metabolic syndrome–associated NAFLD and NAFLD associated with genetic variation in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene? Accumulating evidence indicates that NAFLD is not simply the hepatic manifestation of the metabolic syndrome, but is a pathogenic determinant of the syndrome. Genetic variation within the PNPLA3 gene (e.g., I148M homozygosity) confers a higher risk of developing more severe histological features of NAFLD, but a lower risk of developing metabolic syndrome traits. We suggest that future research is now required to elucidate whether both metabolic syndrome–related NAFLD and PNPLA3-related NAFLD produce the same risk of developing extrahepatic complications. the assertion that the conventional paradigm of NAFLD representing the mere ‘‘hepatic manifestation’’ of the metabolic syndrome is outdated, and it is now becoming clear that NAFLD is also a pathogenic determinant of metabolic syndrome.1 With regard to this evolving concept, there is a growing body of evidence strongly supporting the notion that NAFLD precedes the development of T2DM and metabolic syndrome.4 There are now approximately 30 longitudinal, retrospective, and prospective studies that provide strong evidence for NAFLD as an independent predictor of the future development of T2DM.4–15 Moreover, more than a dozen of longitudinal studies with a relatively long duration of follow-up reported that NAFLD, as diagnosed by either ultrasonography or biochemistry, is independently associated with an increased incidence of several components of metabolic syndrome, not only of T2DM.4,16–24 Amongst the prospective studies that have used ultrasonography to diagnose NAFLD and that have assessed the risk of developing T2DM, the risk of T2DM varied markedly from an approximately 60% increase to approximately a sixfold increase in risk.7–15 This wide interstudy variation in T2DM risk might reflect differences in NAFLD severity, as the study by Park et al.13 showed that the incidence rate of
Introduction
N
onalcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease of this decade, which is in part correlated with the global epidemic of obesity, the metabolic syndrome, and type 2 diabetes mellitus (T2DM).1,2 Cardiovascular disease (CVD) is the leading cause of death in NAFLD,3 followed by malignancy and liver failure, all of which are predominantly due to the progressive form of NAFLD, namely nonalcoholic steatohepatitis.1 The last few years have seen advances in many aspects of NAFLD, including molecular mechanisms involved in hepatic lipotoxicity and insulin resistance (IR), mechanisms of CVD risk, identification of novel gene associations, noninvasive assessment, and therapeutic agents. Here, we highlight some of these findings that are likely to have a major influence on the field.
Does NAFLD Predict the Development of T2DM and Metabolic Syndrome? NAFLD is now increasingly diagnosed in individuals who do not have T2DM or metabolic syndrome. This supports
1 Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University and Azienda Ospedaliera Universitaria Integrata of Verona, Verona, Italy. 2 Nutrition and Metabolism, Faculty of Medicine, University of Southampton, Southampton, United Kingdom. 3 Southampton National Institute for Health Research Biomedical Research Centre, University Hospital Southampton, Southampton, United Kingdom.
1
2
T2DM increased sharply according to the ultrasonographic severity of NAFLD at baseline (normal, 7%; mild, 9.8%; moderate-to-severe, 17.8%). Even after adjustment for multiple confounders, the hazard ratios (HRs) for T2DM development were higher in the mild-NAFLD group [adjusted HR, 1.09; 95% confidence interval (CI) 0.8–1.5] and in the moderate-to-severe-NAFLD group (adjusted HR, 1.73; 95% CI 1.0–3.0) compared with the no-NAFLD group.13 Of note, Sung et al. have investigated the impact of abdominal obesity, IR, and hepatic steatosis on the risk of T2DM at the 5-year follow-up in a cohort of over 12,000 Korean individuals.14 These data showed that each of these three risk factors was associated with T2DM risk, and each risk factor was associated with an approximate doubling of the T2DM risk after adjustment for other established risk factors. Notably, in the same observational cohort of individuals, the authors examined the impact of resolution of hepatic steatosis on the risk of T2DM at the 5-year followup to establish whether NAFLD improvement was associated with T2DM risk reduction. These data demonstrated that there was a significant T2DM risk reduction in those subjects in whom hepatic steatosis on ultrasonography resolved over time. In particular, in those subjects, risk of T2DM decreased to the background risk of someone who had never had hepatic steatosis.15 Conversely, the individuals in whom the severity of hepatic steatosis worsened over 5 years showed a marked increase in T2DM risk [adjusted odds ratio (OR) = 6.13; 95% CI 2.5–14.6 compared with the risk in people with resolution of hepatic steatosis], further supporting the notion that more severe forms of NAFLD are associated with a greater risk of T2DM.15 From these published data it is important to understand how mechanistically pre-existent NAFLD may lead to the development of T2DM and metabolic syndrome. With the exception of cases where NAFLD results from either familial hypobetalipoproteinemia or patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms in which NAFLD is usually dissociated from IR,25,26 NAFLD directly causes hepatic IR in most cases. Molecular and genetic studies have recently supported the view that the pathogenetic mechanisms contributing to intrahepatocytic lipid compartmentation are determinants of whether fatty liver is or is not associated with IR and metabolic syndrome.27 Proposed mechanisms by which NAFLD causes hepatic IR implicate various lipid species, inflammatory signaling, and other cellular modifications.27 Experimental studies have elucidated a key role for hepatic diacylglycerol activation of protein kinase Ce (PKCe) in triggering hepatic IR.28 Alterations in the regulation of intrahepatic lipid droplet metabolism influence the intracellular compartmentation of diacylglycerol, which dictates whether or not PKCe will translocate to the plasma membrane so promoting lipotoxicity and hepatic IR.27,28
Are There Differences Between Metabolic Syndrome–Related NAFLD and PNPLA3-Related NAFLD? Although, as discussed above, NAFLD is strongly associated with metabolic syndrome features,1,2 there are some people who develop NAFLD but do not have metabolic syndrome.29 This suggests that other factors besides metabolic syndrome– specific mechanisms are involved in NAFLD pathogenesis.
TARGHER AND BYRNE
To date, the strongest evidence that any single gene may cause NAFLD, independently of metabolic syndrome, is with a specific polymorphism in the PNPLA3 gene. Increased hepatic fat content30,31 and, in contrast to metabolic syndrome, low circulating serum triglycerides (TG) levels and a dissociation between hepatic steatosis and IR26,32 have been described with the GG allele (I148M) gene variant of PNPLA3 in people with NAFLD. Although there is modest attenuation of metabolic syndrome features, such as improved IR and decreased TG levels, with the GG genotype, it is uncertain whether this may also affect risk of CVD with NAFLD. Perhaps surprisingly, it was recently reported that in patients with NAFLD there was a greater prevalence of increased carotid intima media thickness among those with the GG genotype compared to those with the combined CG + CC genotypes.33 However, further studies are needed to elucidate this issue and determine whether the NAFLD phenotype occurring with the GG allele gene variant is associated with higher or lower risk of CVD. The precise function of PNPLA3 is uncertain, but both hepatic lipogenic and lipolytic roles have been proposed.34 PNPLA3 has acyltransferase activity, converting lysophosphatidic acid to phosphatidic acid within the triacylglycerol synthesis pathway, and also has triacylglycerol hydrolysis activity. It has been suggested that homozygosity of a G / C substitution at position 148 by changing an isoleucine to a methionine increases lipogenic activity and decreases hydrolytic activity. However, elucidating the precise function of hepatic PNPLA3 in NAFLD and investigating the influence of I148M has proved difficult. Until recently, animal studies have also provided limited insight. Very recent work with the I148M PNPLA3 knockin mice has shown that, although these mice have normal liver fat levels on a chow diet, when fed a high-sucrose diet, their liver fat levels increased two- to three-fold.35 The increased liver fat content in these knockin mice was also accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 mRNA.35 These data provide the first direct evidence that physiological expression of the PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is very important and associated with the accumulation of lipid in the liver. The strength of effect of the I148M variant on NAFLD disease severity across different populations has been analyzed in a systematic review and meta-analysis of 16 studies.36 In this analysis, homozygosity for I148M was associated with *75% higher hepatic fat content compared with the CC variant. The I148M GG variant was also associated with more severe NAFLD. For example, GG homozygotes had higher serum aminotransferase levels and an approximately three-fold greater risk of hepatic necroinflammation and fibrosis when compared with CC homozygous individuals. Evaluation of the risk associated with heterozygosity for the I148M variant suggested that the additive genetic model best explained the effect of I148M on the susceptibility to develop NAFLD. Nevertheless, and surprisingly, carrying two G alleles did not seem to increase the risk of severe histological features. Interestingly, metaregression showed an inverse association between male sex and the effect of I148M on hepatic fat content,36 suggesting that there may be dissimilar influences of I148M in men and women.
METABOLIC SYNDROME AND NAFLD
Although decreased serum TG levels32 have been described with the GG variant, Qiao et al. showed in an animal model that PNPLA3 increased lipogenesis, inducing hepatic verylow-density lipoprotein (VLDL)–TG accumulation that, in turn, may be secreted into serum, thereby increasing serum TG levels.37 The mechanisms by which PNPLA3 regulates VLDL assembly and secretion are poorly understood. PNPLA3 might alter the availability of hepatic lipid for VLDL assembly and secretion, thereby affecting serum TG concentrations. Similarly by regulating hepatic lipolytic activity, PNPLA3 might alter the availability of hepatic TG for VLDL assembly and secretion, thereby also affecting serum TG levels. There is some evidence that the PNPLA3 I148M GG genotype facilitates incorporation into liver lipid droplets of different types of fatty acid (e.g., palmitoleic acid), thereby altering the fatty acid composition of the lipid droplets.38 Whether the PNPLA3 genotype influences the fluxes of hepatic fatty acids to affect liver fat and VLDL assembly and secretion in NAFLD is uncertain. Recently, Nobili et al. showed that the PNPLA3 GG genotype diminished the beneficial effect of the long-chain omega-3 polyunsaturated fatty acid docosahexanoic acid (DHA) treatment on liver disease end points in children with NAFLD.39 DHA and eicosapentaenoic acid (EPA) are known to exert hypotriglyceridemic effects,40 and we have recently also shown a benefit of high tissue enrichment with DHA with omega-3 fatty acid treatment to decrease hepatic fat content in NAFLD.41 The hypotriglyceridemic effects of omega-3 fatty acids have also been demonstrated in NAFLD patients,42 and it is known that omega-3 fatty acids reduce hepatic VLDL–TG secretion via apolipoprotein B100 degradation,43 downregulation of de novo TG synthesis,44 enhancement of liver b-oxidation, and reduction of adipose tissue inflammation, thus reducing plasma nonesterified fatty acid delivery from adipose tissue to the liver.45 We suggest further work is needed to understand how PNPLA3 I148M modifies VLDL assembly (and/or secretion) and how PNPLA3 I148M modifies fluxes of omega-3 fatty acids in particular to affect liver disease severity and to affect metabolic syndrome features, such as increased serum TG concentrations in NAFLD.
Conclusions The current evidence supports a key role of NAFLD in the pathogenesis of hepatic IR and T2DM. NAFLD may also have relevant clinical implications for diagnosing, preventing, and treating the metabolic syndrome. Additionally, we believe that considerable attention should be paid to the development of NAFLD in the absence of metabolic syndrome and in the absence of overweight/obesity (i.e., the so called ‘‘lean’’ NAFLD). A key unanswered question is whether ‘‘lean’’ NAFLD and PNPLA3-related NAFLD carry the same risk as NAFLD occurring with metabolic syndrome for the development of extrahepatic (CVD and noncardiovascular) complications. Future well-designed prospective studies will be needed to answer this question.
Acknowledgments G.T. is supported in part by grants from the University School of Medicine of Verona. C.D.B. is supported in part by the Southampton National Institute for Health Research Biomedical Research Centre.
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Author Disclosure Statement No competing financial interests exist.
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Address correspondence to: Prof. Giovanni Targher, MD Division of Endocrinology, Diabetes and Metabolism Department of Medicine University and Azienda Ospedaliera Universitaria Integrata Piazzale Stefani, 1 37126 Verona Italy E-mail: [email protected]
