Synergy between NAFLD and AFLD and potential biomarkers

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Synergy between NAFLD and AFLD and potential biomarkers夽 Raj Lakshman ∗, Ruchi Shah , Karina Reyes-Gordillo , Ravi Varatharajalu Lipid Research Laboratory, VA Medical Center and Department of Biochemistry and Molecular Medicine, The George Washington University, 50 Irving Street, Washington, DC 20422, NW, USA

Summary Fatty liver (hepatosteatosis) is the earliest abnormality in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD) due either to metabolic risk factors associated with insulin resistance and/or metabolic syndrome in the absence of alcohol consumption or to chronic alcohol abuse. When unchecked, both NAFLD and AFLD lead to steatohepatitis, fibrosis, cirrhosis, hepatocellular carcinoma (HCC) and eventual death. A number of common mechanisms contribute to the above various stages of hepatocyte injury, including lipotoxicity, endotoxin release, oxidative and ER stress leading to increased pro-inflammatory cytokines that stimulate hepatic fibrogenesis and cirrhosis by activating the quiescent hepatic stellate cells (HSC) into myofibroblasts. Significantly, patients with either NAFLD or AFLD respond favorably to early treatment modalities to reduce hepatic fat accumulation and consequent increased inflammatory signalling and activation of hepatic stellate cells. Although the pathogenic pathways associated with NAFLD and AFLD are seemingly similar, differentiation of the molecular mechanism/s of the pathogenesis of these liver diseases is critical in identifying the unique molecular signatures, especially in the early diagnosis of NAFLD and AFLD. Current clinical practice requires the invasive biopsy procedure for the conclusive diagnosis of NAFLD and AFLD. Micro RNAs (miRNAs) are ∼22 nucleotide non-coding sequences that bind to the 3 -untranslated region of target transcripts and regulate gene expression by degradation of target mRNAs or inhibition of translation. Emerging studies may prove to establish miRNAs as excellent non-invasive tools for the early diagnosis of various stages of liver diseases. © 2015 Published by Elsevier Masson SAS.

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This article is part of the special issue ‘‘Alcohol, Virus and Steatosis evolving to cancer’’ featuring the conference papers of the 10th International Symposium organized by the Brazilian Society of Hepatology in São Paulo, Brazil, September 30th—October 1st, 2015. ∗ Corresponding author. Tel.: +1 202 745 8330; fax: +1 202 462 2006. E-mail address: [email protected] (R. Lakshman). http://dx.doi.org/10.1016/j.clinre.2015.05.007 2210-7401/© 2015 Published by Elsevier Masson SAS.

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Introduction Non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD) are widely prevalent in a large population worldwide [1,2]. While NAFLD is due to risk factors, such as central adiposity, dyslipidemia, hypertension, insulin resistance (IR), type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS); AFLD is due to chronic alcohol abuse that may or may not be superimposed with the above risk factors. Left unchecked both NAFLD and AFLD may further regress into developing non-alcoholic or alcoholic steatohepatitis (NASH/ASH) characterized by hepatocyte inflammation with or without fibrosis [3,4]. NASH/ASH could lead to hepatic fibrosis or cirrhosis [5], and eventually, even hepatocellular carcinoma (HCC) and death [6,7]. Thus, as a result of unhealthy eating habits, sedentary lifestyles superimposed with or without alcohol abuse, there has been an exponential increase in the obese population globally over the past few decades. Consequently, early diagnosis and the causes for the incidence of NAFLD and AFLD are essential in devising proper therapeutic interventions in the treatment of various liver diseases throughout the world.

Regulation of hepatosteatosis and lipotoxicity The liver plays a major role in lipid and carbohydrate metabolism and the pathogeneses of NAFLD and AFLD is predominantly due to impaired lipid and carbohydrate metabolism and consequent aberrant homeostasis of intermediary metabolism [8]. Hepatosteatosis is the result of imbalance between hepatic lipid synthesis and degradation [9]. Fat synthesis is regulated by lipogenic genes via sterol regulatory element binding protein C (SREBP1c), whereas fat oxidation is regulated by fatty acid oxidation genes via peroxisome proliferator-activated receptor ␣ (PPAR␣). Significantly, SREBP1c and PPAR␣ are tightly controlled by peroxisome proliferator-activated receptor gamma coactivator beta (PGC1␤) and peroxisome proliferator-activated receptor gamma coactivator alpha (PGC1␣), respectively [10]. Silence regulator gene, sirtuin 1 (SIRT1) and histone acetyltransferases (HAT) play dynamic roles in regulating the functional forms of SREBPs and PGC1␣. Thus, SIRT1 destabilizes SREBPs by deacetylation while HAT stabilizes SREBP1c by acetylation. On the other hand, SIRT1 activates PGC1␣ by deacetylation while HAT inactivates PGC1␣ by acetylation. Caloric restriction and low fat activate SIRT1, whereas caloric excess and high-fat activate P38 MAPK by phosphorylation, which inactivates PGC1␣ or stabilizes SREBP1c by activating HAT. Thus, activation/inactivation of PGC1␤, in concert with SREBP1c, would regulate lipid synthesis while activation/inactivation of PGC1␣, in concert with PPAR␣, would regulate lipid oxidation. Lipogenic and lipid oxidation pathways are under tight dietary and hormonal regulation. There are parallel reciprocal responses in the positive direction by insulin and in the negative direction by glucagon, the two opposing pancreatic hormones. The plasma concentrations of these hormones also vary reciprocally in the starved versus the fed condition. Fittingly, this reciprocal complex regulation of lipid metabolism places adenosine 5 -adenosine monophosphate (AMP) — activated protein kinase (AMPK) in a dynamic

role, regulating a multitude of critical pathways in lipid metabolism. AMPK is a metabolic, stress-sensing enzyme that is activated by caloric restriction [high AMP:adenosine tri-phosphate (ATP) ratio] and inhibited by high caloric intake (low AMP:ATP ratio). AMPK is activated by phosphorylation, which then inactivates the rate limiting lipogenic enzyme, acetyl CoA carboxylase (ACC), leading to decreased synthesis of malonyl CoA and long-chain fatty acids and their subsequent esterification to form triacylglycerol. Moreover, high-fat diet has a feedback inhibition of the lipogenic pathway. It is fitting that a high-fat — low-carbohydrate diet blocks lipid synthesis, whereas a fat-free — highcarbohydrate diet stimulates lipid synthesis by inactivating AMPK, with associated activation of ACC. As a result, there would be reciprocal changes in cellular malonyl CoA and long-chain acyl-CoAs. The above-described two metabolites play important roles in regulating the lipogenic and fatty acid degradation pathways. Thus, malonyl CoA, the key intermediate in lipid synthesis, is a potent inhibitor of the transport of long-chain fatty acids into the mitochondria, an obligatory step for intra-mitochondrial fatty acid oxidation [11]. In contrast, long-chain acyl-CoAs, the product of lipid synthesis, are potent inhibitors of ACC, resulting in low malonyl CoA generation [12]. Therefore, the critical branch point that determines the fate of newly synthesized long-chain fatty acids is whether they are directed toward triacylglycerol synthesis in the extra-mitochondrial compartment or toward fatty acid oxidation in the mitochondria. The cellular relative concentrations of long-chain acyl-CoAs versus malonyl CoA delicately control this balance. Thus, one could expect the lipogenic pathway to be blocked with a low malonyl CoA:long-chain acyl-CoAs ratio, as found during starvation/caloric restriction, whereas the mitochondrial pathway should be blocked by a high malonyl CoA:long-chain acyl-CoAs ratio, as encountered during increased caloric intake, obesity, and metabolic syndrome. These aspects have been reviewed [9]. Based on all these, clearly it is not the stored triglyceride per se, but the increased free fatty acids that cause lipotoxicity due to the inability of the liver to oxidize the excess fatty acids.

Oxidative and ER stress Increased generation of reactive oxygen species (ROS) occurs in both NAFLD and AFLD essentially due to either increased hepatic lipid oxidation in various intra-cellular compartments, such as mitochondria, peroxisomes, and the endoplasmic reticulum or ethanol metabolism via Cyp2E1, respectively [13,14]. This leads to extensive damage to liver cell and its organelles unless the excess ROS is promptly quenched by endogenous antioxidant systems. This may even lead to DNA mutation, activation of redox-sensitive transcription factors, such as nuclear factor receptor kappa B (NF␬B) [15,16] that controls the synthesis of inflammatory mediators, such as tumor necrosis factor alpha (TNF␣) and other pro-inflammatory cytokines [17—19], which may cause hepatocyte death receptor signaling via autocrine effects [20,21] or promote inflammatory and fibrogenic responses via paracrine activation of macrophages, endothelial cells and HSC [22—25]. However, clinical trials with antioxidants have not yielded consistent positive results in the treatment

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Synergy between NAFLD and AFLD and potential biomarkers Table 1

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Potential miRNA biomarkers for various stages of liver diseases.

Disease state

NAFLD/NASH AFLD/ASH Fibrosis Cirrhosis HCC AFLD/ASH (Gut) IR/T2DM

Candidate miRNA marker

References

Upregulated

Downregulated

34a, 122 217 34a, 122 34a, 122

let7d

212 146a

of NASH/ASH. Impaired trafficking and accumulation of various proteins in ER leads to ER stress resulting in unfolded protein response [26,27], and impaired protein synthesis in ER membranes and its associated factors [28,29]. This causes autophagic response by the cell for the disposal of the retained proteins. Naturally, ER stress affects lipid homeostasis, and thus seems to be the major mechanism of hepatotoxicity in both NAFLD and AFLD [30].

Pro-inflammatory cytokines Both NAFLD and AFLD are characterized by increased expression and secretion of pro-inflammatory cytokines, particularly TNF␣, interleukin (IL)-1␤, IL-6 and IL-8 [31,32]. Almost all types of cells, such as hepatocytes, cholangiocytes, macrophages, HSC, endothelial cells, and adipocytes are responsible for generating these cytokines when subjected to oxidative stress. Significantly, various animal models of NAFLD/AFLD respond favorably with respect to steatohepatitis when various modes of TNF␣ inhibition are achieved [33,34]. TNF␣ is known to inhibit the expression of adiponectin that protects against liver injury [35], leading to free fatty acid accumulation and consequent lipotoxicity. Furthermore, TNF␣ promotes mitochondrial ROS production and mitochondrial membrane transition resulting in oxidant and apoptotic stress [22,36]. TNF␣ also upregulates downstream kinases that are involved in insulin signalling, thereby causing insulin resistance and hyperinsulinemia, which profoundly influence lipid and carbohydrate metabolism [22,37]. Apart from these actions, TNF␣ induces IL-8 and other chemokines, which, in turn, recruit various inflammatory cells into the liver [38]. Unfortunately, because of exorbitant costs of specific TNF␣ antagonists and the possibility of associated potential toxicity, such specific TNF␣ antagonists are yet to be evaluated in the treatment of NASH/ASH patients.

Micro RNAs and NAFLD versus AFLD Micro RNAs (miR) are ∼22 nucleotide non-coding RNAs that constitute silencers of target gene expression [39]. miRs are generated from highly structured primary (pri-miRNAs) transcripts of RNA Pol II genes by two-step processing events involving RNase III type nucleases. Pri-miRNA transcripts are processed in the nucleus by RNase III type endonuclease,

19b, 132, 29a 29a 16, 141

[40—42] [43] [40,41,44—46] [41—46] [47,48] [49] [50]

Drosha into precursor (pre-miRNA) and exported to the cytoplasm by exportin 5 to be secondarily processed into miRNA duplexes by the cytoplasmic RNAse type III Dicer. The resulting miRNA duplexes are incorporated into the RNA-Induced Silencing Complex (RISC) where one of the miRNA strands, the ‘‘passenger’’ is degraded, while the ‘‘guide’’ strand complementary to the target mRNA, is guided to the target sequence to either degrade (in case of perfect base complementarity) or to block translation (in case of imperfect sequence complementarity, between the 5 -nucleotides 2—8 of miRNA and the 3 -untranslated region (3 -UTR) of the target mRNA). Emerging evidences suggest that aberrant expression of miRs contributes to the development of both NAFLD and AFLD. Table 1 below summarizes, albeit not exhaustively comprehensive, the most significant miRNA signatures that characterize NAFLD, AFLD, NASH, ASH, fibrosis, cirrhosis and HCC with the relevant references. Thus, miR-122 serves as a key regulator of the metabolism of glucose and lipids in adult livers and has been suggested to be a predictive circulating marker of hepatic fibrosis in NAFLD patients [40]. Overexpression of miR-34a, a transcriptional target of p53, and suppression of let7d, a micro RNA precursor, are involved in the pathogenesis of NAFLD [41,42], whereas miR-217 overexpression leads to hepatosteatosis in alcoholic liver [43]. The mechanism of action(s) of both of the miRNAs seems to involve repression of SIRT1, resulting in the inhibition of fatty acid oxidation, and inactivation of AMPK, which in turn upregulates fat synthesis by activating ACC, the rate limiting step of fatty acid synthesis. Significantly, miR-19b has been shown to be specifically downregulated in fibrotic liver, presumably by upregulating TGF␤ and fibrogenic pathway [44], while alcohol abuse mediated miR-132 suppression specifically upregulates MeCP2, the epigenetic promoter of fibrogenic pathway [45]. On the other hand, miR-29a has been shown to be significantly downregulated in fibrotic/cirrhotic livers compared to non-fibrotic livers [46], whereas miR-16 and miR-141 are upregulated in HCC [47,48]. Tang et al. [49] have reported that alcohol-induced overexpression of miR-212 results in gut leakiness, a key factor in alcoholic liver disease by downregulation of tight junctions. Finally, with respect to T2DM, the miR-146a expression is significantly decreased in peripheral blood mononuclear cells in these patients associated with insulin resistance and poor glyceamic control [50].

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Figure 1 Synergy between NAFLD and AFLD during various stages of liver diseases. NAFLD and AFLD have divergent pathogenic origins consisting of either insulin resistance (IR), type 2 diabetes mellitus (T2DM) and metabolic syndrome, or alcohol overconsumption, respectively, but share common downstream molecular pathogenic pathways that involve lipotoxicity, endotoxin release, leptin, oxidative stress, and ER stress, which lead to an inflammatory response caused by the activation of NF␬B signaling resulting in pro-inflammatory cytokines induction in the hepatic Kupffer cells. Moreover, they also act as fibrogenic stimuli that lead to the transdifferentiation of quiescent HSC to activated HSC by suppression of adipogenic PPAR and upregulation of fibrogenic MeCP2, and TGF␤ signaling pathways resulting in extensive hepatic fibrogenesis. Various micro RNAs play diverse roles in regulating the different stages of liver diseases.

Summary In summary, as depicted in Fig. 1, while NAFLD, AFLD and the sequelae of this complex disease have divergent pathogenic origins, they share common downstream molecular pathogenic pathways of involving lipotoxicity, endotoxin release, oxidative stress, and ER stress, which lead to both inflammatory and fibrogenic stimuli. These, in turn, upregulate:

• NF␬B, and pro-inflammatory cytokines in the hepatic Kupffer cells; • transdifferentiate quiescent HSC to activated HSC by suppression of adipogenic PPAR and upregulation of fibrogenic MeCP2, and TGF␤ signaling pathways resulting in extensive hepatic fibrogenesis.

When unchecked, the fibrotic liver deteriorates into cirrhosis, and in some instances, into HCC, and eventually death. Therefore, early accurate diagnosis of the various stages of these liver diseases is clinically very important. Current clinical practice requires the invasive biopsy procedure for the conclusive diagnosis of NAFLD and AFLD. miRs are ∼22 nucleotide non-coding sequences that bind to the 3 -untranslated region of target transcripts and regulate gene expression by degradation of target mRNAs or inhibition of translation. Emerging studies seem to establish miRs as excellent non-invasive tools for the early diagnosis and treatment of various stages of liver diseases.

Implications and future directions Intensive research by investigators worldwide in the elucidation of the molecular mechanisms for the pathogenesis

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of various stages of NAFLD and AFLD has led to a better understanding of these complex liver diseases. However, morbidity and mortality due to chronic liver diseases is staggering throughout the world due to increasingly poor dietary habits, lifestyles, T2DM, metabolic syndrome, and/or alcohol abuse. Therefore, early diagnosis and monitoring of the various stages of liver diseases is of utmost clinical importance to effectively treat such patients. While, the existing methods are invasive and possibly may cause harm to these patients, epigenetic non-invasive methods have offered a novel perspective not only into the identification of pathogenesis of the disease, but also to potentially develop accurate early diagnostic biomarkers, and therapy.

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Disclosure of interest The authors have not supplied their declaration of conflict of interest.

Acknowledgments This work is supported by the NIH grants RO1 AA020720 (MRL), RO1 AA009231 (MRL), and R21 AA022205 (MRL).

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