ACHAIKI IATRIKI | 2020; 39(2): 94–106
Review
Sebastien Filippas-Ntekouan, Ilias Tsiakas, Angelos Liontos, Ekaterini Panteli, George Kalambokis, Haralampos Milionis
Department of Internal Medicine, School of Medicine, University of Ioannina, Ioannina, Greece
Received: 17 April 2020; Accepted: 25 June 2020
Corresponding author: Haralampos Milionis, MD, PhD, Professor of Internal Medicine, Department of Internal Medicine, School of Medicine, University of Ioannina, 45110 Ioannina, Greece, E-mail: hmilioni@uoi.gr, tel: +302651007500
Key words: NAFLD, insulin, insulin resistance
Abstract
Non-Alcoholic Fatty Liver Disease (NAFLD) is considered the leading cause of chronic liver disease and has been associated with cardiovascular disease. Due to the complexity of its pathogenesis, a multiple hit hypothesis has been proposed. Furthermore, NAFLD is associated with both insulin resistance and type 2 diabetes mellitus. Herein, we provide an overview of the effect of insulin and insulin resistance on the pathogenesis of NAFLD.
Introduction
Non-alcoholic fatty liver disease (NAFLD) is characterized by the presence of fat within the hepatic parenchyma, that cannot be attributed to other causes such as viral hepatitis, alcoholic hepatitis, endocrinological conditions or drug intake [1]. For many years, NAFLD has been considered the hepatic manifestation of the metabolic syndrome, and now constitutes a leading cause of chronic liver disease in many parts of the world [1]. It has been shown that up to 85% of subjects with NAFLD compared to 30% in controls are insulin resistant and have abnormal glucose metabolism, i.e., prediabetes or undiagnosed diabetes mellitus type II (T2DM) [2]. NAFLD prevalence in the general population is about 30-40% in men and 15-20% in women, which means that it approximately affects one out of four persons [3]; in type 2 diabetics its prevalence has been reported to be as high as 70% [4]. Although NAFLD has been traditionally considered a disease of the obese, variants of NAFLD in lean individuals have also been described. In this regard, it is prudent to exclude other causes of ectopic liver lipid accumulation such as genetic diseases (lipodystrophies, abetalipoproteinemias, Wilson’s disease), infectious causes (particularly HCV-genotype 3), inflammatory causes (celiac disease), drugs (amiodarone, and others), and others, before attributing fatty liver to an underlying dysmetabolic cause [5].
The importance of diagnosing and treating NAFLD is demonstrated by a meta-analysis, which shows that the presence of NAFLD is associated with a twofold risk of type 2 diabetes mellitus (T2DM) and an increase in overall mortality by 57%, mainly due to liver-related and cardiovascular causes [6].
Insulin is an anabolic hormone which is secreted by the beta cells of the pancreatic islets in response to high serum glucose levels. The liver actions of insulin include: inhibition of hepatic gluconeogenesis and increase of glucogen production and storage [7]. Furthermore, insulin decreases the rate of lipolysis and increases the rate of de novo lipogenesis (DNL), which plays a major role in the development of NAFLD. Hepatic lipid accumulation in NAFLD has been proven to lead to insulin resistance [8], while at the same time elevated insulin levels seem to contribute to the pathogenesis of NAFLD, thus completing the vicious circle. Furthermore, insulin resistance has been shown to be an important factor independently contributing to the progression of NAFLD to NASH [9]. The mechanisms underlying the pathogenesis of NAFLD are quite well described and consist of a sequence of multiple “hits” which include ectopic lipid accumulation, insulin resistance, hyperinsulinemia and oxidative stress, while the gut microbiota and various adipokines play a significant role [10-12].
In this review, we will discuss the altered metabolic pathways which contribute to the development of NAFLD. Specifically, de novo lipogenesis, which is the pathway which synthesizes fatty acids, and its regulation by insulin will be discussed. Furthermore, as peroxisome proliferator activated receptors are central to the regulation of metabolism, their contribution to the development of NAFLD will be addressed. Fox a2 is another pathway which mediates lipid metabolism in the fasting state and will also be discussed.
De novo lipogenesis and its role in NAFLD
De novo lipogenesis (DNL) is the metabolic pathway which synthesizes fatty acids from a carbohydrate substrate [13]. Through glucolysis, acetyl-coA is produced from glucose and serves as the initiator of DNL [14]. The production of malonyl-coA from acetyl-coA is a crucial step which is catalyzed by the enzyme acetyl-coA carboxylase (ACC). Malonyl-coA is transferred to the prosthetic phosphopantetheine group of acyl-carrier protein (ACP), a domain of the type 1 fatty acid synthase (FAS) [15] with subsequent release of coenzyme A carrier catalyzed by the malonyl/acetyl transferase [16]. The ACP-bound malonyl moiety acts as the additive monomer for the elongation of the substrate acyl chain. Initially, this is an acetyl unit bound to the thiol group of cysteine (Cys161) at the β-ketoacyl synthase active site [17]. The malonyl moiety undergoes decarboxylative condensation with an acetyl moiety. ACP is subsequently bound to a β-ketoacyl intermediate. ACP shuttles the β-ketoacyl intermediate to the NADPH-dependent β-keto reductase (KR) active site [18]. The ketone of the β-carbon is reduced, generating a hydroxyl group. This is followed by sequential dehydration, by the dehydratase active site, and further reduction by the NADPH-dependent enoyl-reductase [19]. This generates a saturated acyl chain elongated by two carbon groups, which can act as the substrate for the next round of elongation as it binds the thiol-group of the cysteine to the catalytic site of β-ketoacyl synthase (KS). The elongation stops at the 16- or 18-carbon stage and palmitic or stearic acid is released from ACP via activity of the thioesterase (TE) domain of FAS [20, 21]. The main product of DNL has been proven to be palmitate in rat liver [20, 22] (Figure 1).
Figure 1. De novo lipogenesis. Abbreviations: ACC, acetyl-coA carboxylase; ACP, acyl-carrier protein; coA, coenzyme A; DH, dehydratase; DNL, De novo lipogenesis; EnR, enoyl-reductase; FAS, fatty acid synthase; KR, β-ketoreductase; KS, β-ketoacyl synthase; NADP+ , nicotine adenine dinucleotide phosphate; NADPH, reduced form of NADP+ ; MAT, malonyl/acetyl transferase; TE, thioesterase.
Τhe main substrate for DNL is glucose. Glucose, but also fructose, can be used to produce dihydroxyacetone phosphate which is then turned into glycerol 3 phosphate [23]. Glycerol phosphate acyl-transferase then incorporates fatty acids, synthesized by DNL, to produce lysophosphatidic acid which is used to produce triglycerides [24].
Studies have proven that 59% of the accumulated liver fat in NAFLD comes from free fatty acid flux due to excess adipose tissue, 26% comes from DNL and 15% comes from excess dietary intake [25]. Knowing that DNL produces saturated fatty acids and that people with NAFLD present higher levels of saturated fatty acids, we can conclude that DNL is overworking in NAFLD [26].
Regulation of DNL and the role of insulin
The transcriptional regulation of DNL has two major pathways, the sterol regulatory binding protein 1c (SREBP-1c) and the carbohydrate response element binding protein (ChREBP). SREBP-1c is activated by insulin signaling, while ChREBP is activated by serum glucose concentrations [27, 28]. The activation of these proteins upregulates crucial enzymes, such as ACC and FAS [25, 29]. SREBP1-c is the main regulator of DNL in the liver. SREBP1-c is located in the Golgi membrane and interacts with two membrane proteins of the endoplasmic reticulum, SCAP (SREBP1 cleavage activated protein) and INSIG (insulin induced gene protein) [30]. Phosphorylation events following activation of IRS-1, PI3K, PKB, and mTORC1 by insulin, uncouple SREBP1 from the binding proteins, allowing it to enter the nucleus and activate DNL through transcriptional upregulation of several genes involved in FA synthesis.
It is now known that NAFLD co-exists with insulin resistance and hyperinsulinemia [29]. Furthermore, it has been found that in NAFLD, SREBP-1c is upregulated [31, 32]. The activation of SREBP-1c by excess insulin, causes an increase of lipogenic enzymes, FAS and ACC, leading to an increase in the hepatic biosynthesis of fatty acids and triacylglycerols [33-35].
The question that arises is the following: given that NAFLD is an insulin resistant state, which means that the SREBP-1c pathway should be suppressed, how can its lipogenic enzymes be still upregulated? It is well known that insulin resistance develops in conjunction with lipid metabolism disorders [8, 36-38]. In healthy people, insulin activates SREBP-1c which in turn activates essential lipogenic enzymes, while at the same time insulin inhibits fork head box protein O1 (FOX O1), in an Akt dependent way, which inhibits genes responsible for gluconeogenesis and the increase of fasting serum glucose [39].
In order to fully explain the metabolic features of insulin resistance and hyperinsulinemia, the theory of ‘selective insulin resistance’ has been proposed. According to this, certain insulin activated pathways have become resistant, while at the same time other pathways are still sensitive to insulin signaling. In accordance to this theory, it has been suggested that the FOX O1 pathway, which suppresses hepatic glucose production, is resistant to insulin signaling, while on the other hand, insulin-stimulated de novo lipogenesis is still sensitive (SREBP-1c pathway) [40]. It has been shown that in chronic hyperinsulinemia due to insulin resistance, there are increased levels of SREBP1-c mRNA and precursor protein levels, [41] which means that DNL not only is not suppressed, but is rather increased. This could be explained taking into account that the pathway leading to DNL activation (SREBP-1c), requires fewer activated insulin receptors, while the pathway which inhibits glucose production (FOX-O1) requires a greater number of activated receptors (IRS-1). Moreover, in chronic hyperinsulinemia, the production of insulin receptors is downregulated in hepatocytes. These two findings combined, seem to explain and support the hypothesis of selective insulin resistance, and how DNL is upregulated in hyperinsulinemic states such as in NAFLD [42] (Figure 2).
Figure 2. Selective insulin resistance. In normoinsulinemic states, insulin stimulates insulin receptors and simultaneously inhibits hepatic neoglugenesis and activates De Novo Lipogenesis. In chronic hyperinsulinemic (CHI) states, insulin receptors are downregulated. Furthermore, it has been found that fewer activated insulin receptors are needed to activate DNL, in contrast to inhibiting gluconeogenesis which requires more activated receptors. Given that in CHI insulin receptors are downregulated, hepatic gluconeogenesis is resistant to insulin action while DNL is still sensitive.
Targeting DNL as a treatment target
DNL has been identified as a treatment target for NAFLD/NASH by regulating major transcription factors such as SREBP-1c or ChREBP or by inhibiting specific enzymes. Multiple FXR-activating drugs are in development (stimulation of FXR leads to downregulation of SREBP-1c). Obeticholic acid is the leading drug candidate as a phase 2 trial has showed histological improvement of steatosis and a small improvement of fibrosis in patients treated with obeticholic acid [43]. In addition to targeting the broad transcriptional control of DNL, the sequential contributors to DNL can be individually targeted. Inhibition of ATP citrate lyase (the first enzyme in the DNL cascade) has been proposed as a target, but has only be tested in animal studies [44]. Inhibition of ACC is also being evaluated in ongoing clinical trials. One inhibitor of ACC1/2, firsocostat (GS-0976), induced dose-depended liver fat reduction [45]. Uptake and retention of fructose by hepatocytes may also be a target. Once fructose enters the hepatocyte, it is trapped by phosphorylation and is subjected to further metabolism, especially DNL. One approach of reducing the contribution of fructose to DNL might be with ketohexokinase (fructokinase) inhibitors that block fructose phosphorylation and one is currently being evaluated in a clinical trial [46].
Peroxisome-proliferator activated receptors (PPARs) and the role of insulin (Table 1)
Peroxisome-proliferator activated receptors (PPARs) are nuclear receptors found in many tissues (such as the liver and the adipose tissue) and their main role is to regulate energy production, lipid and glucose metabolism, as well as to regulate cell proliferation [47]. PPARs regulate the expression of genes implicated in carbohydrate, fatty acid and cholesterol metabolism. Furthermore, PPARs regulate genes responsible for cell proliferation, tumorigenesis and inflammation [48]. PPAR-α are found in tissues with high fatty acid oxidation rates, such as the liver, muscles, heart muscle and the kidneys. PPAR-α-deficient mice develop fatty liver in contrast to normal mice [49]. Their activation leads to an increase in fatty acid peroxisomal and mitochondrial oxidation, fatty acid transport and increased ketogenesis [50] through the activation of several enzymes such as carnitine palmitoyl-transferase 1 (CPT-1) [51]. CPT-1 is a regulatory enzyme found in mitochondria, which transfers fatty acids from the cytosol to the mitochondria so that β-oxidation starts [52]. Furthermore, PPAR-α enhances the production of the enzyme FAT/CD36 which is responsible for fatty acid cell uptake [50]. Although seemingly divergent, PPAR-α also control lipogenic enzymes by enhancing SREBP-1 cleavage [53]. In the mouse liver, the SREBP-1c target genes, i.e. FAS, ACC1, and SCD-1, are upregulated by PPAR-α agonists although insulin signaling is still required in order to fully activate DNL [54, 55]. In human primary hepatocytes, PPAR-α agonists, in cooperation with insulin and LXR agonists, induce lipogenic gene expression, such as FAS and ACC1 [56]. The lipogenic product malonyl-coA inhibits the action of CPT-1. In the fasting state, the organism uses fatty acid as energy provider through activation of PPAR-α. On the other hand, in the fed state, PPAR-α agonists change and promote de novo lipogenesis in an insulin dependent manner. Thus, insulin increases lipid accumulation in NAFLD in two ways: firstly, chronic hyperinsulinemia directly upregulates SREBP-1 and secondly, indirectly, through DNL activation and malonyl-coA production, which inhibits CPT-1 [57]. PPAR-α activation through fenofibrate which is commonly used to treat hypertriglyceridemia, has not been shown to be effective. On the other hand, bezafibrate has been proven effective in small studies [58].
PPAR-β/δ are found in the majority of human tissues and their activation leads to a number of different effects including an increase of glucose uptake and use by peripheral tissues, an increase of fatty acid oxidation in the adipose tissue, an amelioration of the lipidemic profile (increase of HDL-CHOL, apo-A1 and decrease of LDL-CHOL) and a protective role against atherosclerosis as well as against NAFLD and its progression to steatohepatitis [59].
Activation of PPAR-β/δ in hepatocytes has resulted in reduced glucose release by the liver leading to better glucose homeostasis and amelioration in insulin resistance [60]. Increased release of fatty acids from adipocytes and glucose from hepatocytes are the major abnormalities of the metabolic syndrome. Treatment of obese animals with a PPAR-β/δ agonist resulted in decreased hepatic glucose output, and increased glucose disposal [61]. These changes were followed by an increase in glucose metabolism via the pentose phosphate pathway leading to increased de novo biosynthesis of fatty acids. Administration of the PPAR-β/δ agonist GW501516 to obese mice resulted in decreased accumulation of triglycerides in adipocytes and hepatocytes [62]. Overall, PPAR-β/δ agonist increases HDL-cholesterol levels, reduces LDL-cholesterol levels, increases fatty acid oxidation in adipose tissue and mitigates blood vessels inflammation, thus representing a potent means of treating metabolic syndrome associated morbidities. The PPAR-δ activating drug, seladelpar, has been evaluated in a phase 2a trial and preliminary results showed improved serum ALT and LDL-cholesterol levels but no improvement in liver fat; liver biopsy results at 52 weeks are not yet available and development of this drug is currently on hold due to safety concerns [63].
PPAR-γ is found mainly in the adipose tissue but also in other tissues such as the large intestine, the liver, the muscles and the immune system. PPAR-γ activation in the adipose tissue promotes lipid accumulation within adipocytes, and the differentiation of mesenchymal cells into adipocytes, thus augmenting adipose tissue lipid storage capacity [64]. In healthy individuals, PPAR-γ is expressed at low levels in the liver [65]. In hepatocytes, PPAR-γ activation increases lipid accumulation by enhancing the production of enzymes responsible for DNL and fatty acid uptake. In hyperinsulinemic states, such as NAFLD, obesity and the metabolic syndrome, PPAR-γ expression in the liver is increased. Activation of PPAR-γ in the liver by excess insulin enhances production of the enzyme fatty acid translocase (FAT/CD36) which acts as a mediator of hepatic fatty acid uptake and increases intrahepatic lipid accumulation [66]. However, PPAR-γ signaling in humans is not correlated with hepatic steatosis [67]Md.. Thiazolinediones (TZDs), an anti-diabetic drug class, are PPAR-γ agonists and insulin-sensitizers widely used in the treatment of T2DM and insulin resistance. Several human studies in patients with insulin resistance who received TZDs, showed that hepatic steatosis was not worsened but, rather, improved [68]. Thus, any PPAR-γ-induced hepatic lipid accumulation in humans treated with TZDs is outweighed by the more prominent beneficial effects on fatty acid storage in the adipose tissue. This can be explained by the fact that PPAR-γ activation increases the number of adipocytes which can secrete various adipokines such as adiponectin [69]. Adiponectin is a known insulin sensitizing factor which ameliorates insulin sensitivity in the liver, while triggering hepatocytes to burn the excess fat [70, 71]. To sum up, PPAR-γ activation in the liver by insulin has been shown to increase hepatic steatosis, thus worsening NAFLD, yet PPAR-γ activation in the organism leads to an improvement in hepatic histology and may be used in the treatment of NAFLD. Pioglitazone is the most widely clinically used PPAR-γ ligand and its benefits in NASH have been demonstrated [72, 73].
Drugs that target more than one PPAR nuclear receptor are also being investigated. Elafibranor, a PPAR-α and PPAR-δ activator has been shown to improve liver histology [74] and this drug is being further evaluated in the large international RESOLVE-IT trial (NCT02704403). Drugs that target both PPAR-α and PPAR-γ have been explored in diabetes, but only saroglitazar continues to be evaluated in NASH with preliminary results showing improved ALT and liver fat content but some weight gain at 16 weeks. Lanifibranor is a pan-PPAR agonist currently being evaluated in a phase 2b study and results are pending.
Fox a2 and the role of insulin
Fox a2 is a key regulator of hepatic lipid metabolism and in the fasting state it induces expression of enzymes involved in fatty acid oxidation, ketogenesis (ketone body production), very low-density lipoprotein (VLDL) secretion, and bile acid metabolism [75, 76]. In the fasting state, glucagon activates Fox a2 increasing the rate of fatty acid oxidation, while in the postprandial state, insulin inhibits its activation in a PI3K/Akt mediated phosphorylation cascade which attenuates Fox a2 actions [77, 78]. Insulin inhibits the transcriptional activity of Fox a2 in the liver not only during feeding, but also in hyperinsulinemic ob/ob or db/db mice and in animals with diet-induced obesity. Expression of constitutively active Fox a2-T156A in diabetic mice normalizes plasma glucose levels, decreases hepatic triglyceride content, increases insulin sensitivity, and elevates hepatic lipid metabolism by activating the expression of genes encoding enzymes involved in mitochondrial b-oxidation and ketogenesis [79]. In summary, in hyperinsulinemic states, excess insulin inhibits Fox a2 which in turn decreases the rate of fatty acid oxidation and contributes to the lipid accumulation observed in NAFLD [80, 81]. (Table 2)
Hepatokines and NAFLD
Fetuin A, also named α2-HS-glycoprotein (AHSG), is secreted primarily by the liver, and by other tissues, including the placenta, adipose tissue and tongue [82, 83]. Fetuin A was first discovered as an inhibitor of insulin receptor tyrosine kinase in the liver and muscles [84]. In 2002, the AHSG gene defected mice were reported to demonstrate improved insulin sensitivity, indicating its role in insulin regulation pathways [85]. Clinically, case control and cohort studies have found that serum fetuin A level is significantly elevated in patients with T2DM, NAFLD and atherosclerosis, which makes fetuin A a potential disease risk marker [86-89]. In mice with hepatic steatosis, upregulated mRNA level of fetuin A was observed in liver tissue. Fetuin A was also found to greatly promote the secretion of pro-inflammatory cytokines in monocytes and adipose tissue and inhibit the expression of an insulin sensitizing hormone, adiponectin [90], Therefore it has been shown that, fetuin A participates in the pathogenesis of NAFLD by inducing insulin resistance and activating inflammatory pathways, acting as a liaison between metabolic dysregulation and inflammatory responses [91].
Fibroblast growth factor 21 (FGF-21) is a 209-amino acid protein mainly secreted by the liver, which can also be detected in the pancreas, the testis and the adipose tissue [92, 93]. FGF-21 serum levels are associated with the development of multiple metabolic diseases, such as obesity, Τ2DM and NAFLD, while FGF-21 levels also correlate with the progression of NAFLD to NASH [94-97]. FGF-21 mediates its effects by binding to its receptor complex, FGF-receptor and the co-receptor β-klotho. FGF-21 acts as an acute insulin sensitizer in multiple ways, such as decreasing insulin levels, increasing the expression of GLUT-1, activating PPAR-γ, inducing the expression of adiponectin, a known insulin sensitizer, while at the same time, FGF-21 has been shown to exert a protective effect on pancreatic β-cells [98]. On this basis, multiple pharmaceutical products aiming to enhance the effect of circulating FGF-21 either by stable FGF-21 analogs or by decreasing FGF-21 breakdown have been tested in mice, although so far, no human clinical trial has been carried out. Interestingly, ER stress is a stimulus for FGF21 release, potentially as a protective response, given that in mouse models, FGF21 has been shown to be protective against ER stress [99]. Ursodeoxycholic acid (UDCA) has not been proven to be beneficial in NASH, but two variants, tauro-UDCA and nor-UDCA that are protective against ER stress are being evaluated in NASH. A recent randomized controlled trial demonstrated that nor UDCA, at a daily dose of 1500 mg for 12 weeks (EudraCT 2013-004605038), induced a small reduction in ALT levels [100].
MicroRNAs in NAFLD
MicroRNAs (miRNAs) are small non-coding RNAs, which can modulate gene expression at the post-transcriptional level by targeting messenger RNAs and inhibiting their translation or promoting their degradation [101]. Various miRNAs have been found to be either increased or decreased in the sera of patients with NAFLD compared to non-NAFLD patients [102, 103]. Among these miRNAs, miR-34a, miR-122, have been most frequently associated with the pathogenesis of NAFLD. MiR34a acts by inhibiting PPAR-a, SREBP-1c and Liver X receptor and in turn leads to dysregulation of hepatic lipid metabolism. Inhibition of miR34a leads to increased PPAR-a expression and restoration of lipid metabolism. miR-122 is the most abundant miRNA in the liver and plays a fundamental role in liver physiology [104-106] and lipid metabolism [107]. miR-122 interacts with multiple important lipogenic factors, such as acetyl coA carboxylase-2 (ACC2) and the sterol regulatory element binding protein (SREBP) [103, 108, 109]. Inhibition of miR-122 in high-fat fed mice has been associated with a significant reduction in hepatic steatosis and plasma cholesterol levels, which was associated with a reduction in hepatic sterol and fatty acid synthesis rates and stimulation of hepatic fatty-acid oxidation mediated by activation of adenosine 5’-monophosphate-activated protein kinase (AMPK) [107].
Extracellular vesicles in NAFLD
Extracellular vesicles (EVs) are submicron membrane-bound structures secreted from different cell types containing a wide variety of bioactive molecules (e.g., proteins, lipids, and nucleic acids (coding and non-coding RNA) and mitochondrial DNA. EVs have important functions in cell-to-cell communication and are found in a wide variety of tissues and body fluids. Data from different diet-induced animal models of NASH have shown that EV concentration increases with disease progression in a time-dependent manner [110-112]. This seems to be a response to the accumulation of toxic lipids and their downstream mediators in the liver, which increase the capacity of hepatocytes to form and release different types of EVs [113, 114]. In vitro treatment of hepatocytes with non-esterified fatty acids evokes the release of EVs containing numerous molecules including C-X-C motif ligand 10 (CXCL10), sphingosine-1-phosphate (S1P), mitochondrial DNA (mtDNA), micro-RNAs, ceramides, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). These molecules may amplify inflammation through multiple mechanisms such as macrophage activation and monocyte chemotaxis, as well as inflammasome activation and modulation of the NF-κB pathway in target cells [114, 115]. In conclusion, EVs have been implicated in the pathogenesis of NAFLD through altered lipid metabolism and immune dysfunction. EVs have also been tested as potential therapeutic agents in animal models of NAFLD. EVs can either be used to alter the expression of various intracellular pathways, or as trojan horses, i.e. EVs can be coupled with an active molecule which can act at a specific tissue in a targeted manner [116]. This is an area of growing interest, and further research is warranted.
Major points of pathophysiology
NAFLD has become the leading cause of chronic liver disease nowadays. Its prevalence is about 20-25% in the general population, so it is of great importance that its pathogenesis is fully understood and described. Although in the past NAFLD was considered the consequence of the metabolic syndrome, nowadays this point of view is obsolete, since it is not so clear which is the first hit: insulin resistance leads to NAFLD, or NAFLD leads to insulin resistance.
The pathogenesis of NAFLD is multifactorial; several factors have been implicated, such as obesity, insulin, adipokines and chronic inflammation, bacterial translocation. Insulin’s actions on promoting hepatic lipid accumulation are shown in (Figure 3). The major players appear to be the hepatic de novo lipogenesis (DNL), PPAR functions and liver lipid oxidation and ketogenesis.
Figure 3. Summary of insulin’s actions in NAFLD. Insulin activates SREBP-1c which in turn activates DNL and produces malonyl-coA. MalonylcoA inhibits β-oxidation by inhibiting the crucial enzyme CPT-1. Also, malonyl-coA is used to synthesize fatty acids. Insulin activates PPAR-γ in the liver which activates the enzyme FAT which increases lipid accumulation. Insulin inhibits fox-a2 which in turn inhibits β-oxidation. All these actions contribute to fatty acid accumulation within the hepatocytes, thus aggravating NAFLD
DNL is the metabolic pathway through which fatty acids are synthesized in the liver, using a carbohydrate substrate (e.g. glucose or fructose). DNL is a useful mechanism in the human organism, since it promotes the production and storing of energy in the form of fatty acids, when an excess of substrate is available. This becomes evident in the postprandial state. In the postprandial state, glucose levels are elevated and in response the beta cells of the islets of Langerhans in the pancreas release insulin. One of the many actions of insulin is to facilitate the entrance of glucose within hepatocytes which means that there is an increased level of DNL substrate within the liver. Insulin also activates SREBP-1 which in turn activates enzymes including FAS and ACC which produce fatty acids. Insulin resistance is present in NAFLD, with hyperinsulinemia being the norm. It has been shown that although insulin’s actions on glucose homeostasis are resistant, insulin’s actions regarding DNL are still sensitive to insulin signaling. Thus, the excess insulin activates overtly DNL which in turn increases the liver content of fatty acids, thus aggravating NAFLD.
PPARs are nuclear receptors found in many tissues which regulate a wide variety of mechanisms. PPAR-α activation leads to increased hepatic lipid oxidation and is the basis for the function of the drug class fibrates (such as fenofibrate) which are primarily used as hypolipidemic drugs. PPAR-β/δ activation ameliorates glucose homeostasis and drugs activating PPAR-β/δ are currently under investigation as potential anti-diabetic agents. Finally, PPAR-γ activation in the liver leads to increased fatty acid uptake as well as increased DNL. PPAR-γ activators such as pioglitazone have been successfully used in the treatment of NAFLD and other insulin-resistant states. Insulin does not seem to have much impact on direct PPAR activation, as PPAR-α and PPAR-β/δ have not been proven to be insulin sensitive. In contrast, PPAR-γ have been shown to be activated by insulin signaling directly or indirectly suggesting that, insulin, increases hepatic fatty acid content in NAFLD in a PPAR-γ dependent way too.
Foxa2 is a regulator of energy homeostasis and is activated by glucagon in the fasting state, while it is inhibited by insulin in the post prandial state. When in the fasting state, glucose levels fall, and the alpha cells of the islets of Langerhans in the pancreas release glucagon, a hormone which opposes insulin effects. Glucagon signaling in the liver is responsible for the depolymerization of glucogen to glucose in order to maintain normal glucose levels; it is also responsible for lipid oxidation and ketogenesis, so that lipids can be used as energy substrate in the tissues, with the exemption of the brain. Insulin acts against these effects, which means that in hyperinsulinemic states, excess insulin inhibits lipid oxidation in the liver, in a Fox a2-dependent way. This leads to decreased clearance of fatty acids from the liver, thus enhancing the lipid accumulation observed in NAFLD.
Concluding remarks
Insulin resistance and consequent hyperinsulinemia:
- upregulates SREBP-1c, thus stimulating hepatic de novo lipogenesis
- enhances PPAR-γ function in the liver, resulting in greater free fatty acids uptake
- inhibits metabolic pathways (Fox a2) and enzymes (CPT-1) linked with hepatic β-oxidation resulting in suppression of β-oxidation and fatty acid accumulation.
Further research is required in order to determine whether targeting the afore-mentioned mechanisms could be of benefit against the progression of NAFLD to NASH and cirrhosis, and/or even help treat obesity-related conditions such as the metabolic syndrome and T2DM.
Conflict of interest disclosure
None.
Declaration of funding sources
None.
Author contribution
Sempastian Filippas-Ntekouan, Angelos Liontos, George Kalambokis, Haralambos Milionis: study conception and design; Sempastian Filippas-Ntekouan, Ilias Tsiakas, Angelos Liontos, Ekaterini Panteli: acquisition of data; Sempastian Filippas-Ntekouan, Ilias Tsiakas, Angelos Liontos, George Kalambokis Haralambos Milionis: analysis and interpretation of data; Sempastian Filippas-Ntekouan, Ilias Tsiakas, Ekaterini Panteli: drafting of manuscript; Angelos Liontos, George Kalambokis, Haralambos Milionis: critical revision.
References
1. Smith BW, Adams LA. Non-alcoholic fatty liver disease. Crit Rev Clin Lab Sci. 2011;48(3):97-113.
2. Targher G, Zoppini G, Day CP. Risk of all-cause and cardiovascular mortality in patients with chronic liver disease. Gut. 2011;60(11):1602-3; author reply 3-4.
3. Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, et al. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology. 2004;40(6):1387-95.
4. Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Ηepatol. 2013 Mar;58(3):593-608.
5. Albhaisi S, Chowdhury A, Sanyal AJ. Non-alcoholic fatty liver disease in lean individuals. JHEP Rep. 2019;1(4):329-41.
6. Musso G, Gambino R, Cassader M, Pagano G. Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity. Ann Med. 2011;43(8):617-49.
7. Owen OE, Reichard GA, Jr., Patel MS, Boden G. Energy metabolism in feasting and fasting. Adv Exp Med Biol. 1979;111:169-88.
8. Samuel VT, Petersen KF, Shulman GI. Lipid-induced insulin resistance: unravelling the mechanism. Lancet. 2010;375(9733):2267-77.
9. Dixon JB, Bhathal PS, O’Brien PE. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology. 2001;121(1):91-100.
10. Day CP, James OF. Steatohepatitis: a tale of two «hits»? Gastroenterology. 1998;114(4):842-5.
11. Stankovic MN, Mladenovic DR, Duricic I, Sobajic SS, Timic J, Jorgacevic B, et al. Time-dependent changes and association between liver free fatty acids, serum lipid profile and histological features in mice model of nonalcoholic fatty liver disease. Arch Med Res. 2014;45(2):116-24.
12. Dowman JK, Tomlinson JW, Newsome PN. Pathogenesis of non-alcoholic fatty liver disease. QJM. 2010;103(2):71-83.
13. Sanders FW, Griffin JL. De novo lipogenesis in the liver in health and disease: more than just a shunting yard for glucose. Biol Rev Camb Philos Soc. 2016;91(2):452-68.
14. Smith S, Tsai SC. The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat Prod Rep. 2007;24(5):1041-72.
15. Brindley DN, Matsumura S, Bloch K. Mycobacterium phlei Fatty Acid Synthetase—A Bacterial Multienzyme Complex. Nature. 1969;224(5220):666-9.
16. Mikkelsen J, Højrup P, Rasmussen MM, Roepstorff P, Knudsen J. Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase. Biochem J. 1985;227(1):21-7.
17. Witkowski A, Joshi AK, Smith S. Characterization of the interthiol acyltransferase reaction catalyzed by the beta-ketoacyl synthase domain of the animal fatty acid synthase. Biochemistry. 1997;36(51):16338-44.
18. von Wettstein-Knowles P, Olsen JG, McGuire KA, Henriksen A. Fatty acid synthesis. FEBS J. 2006;273(4):695-710.
19. Wakil SJ. Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry. 1989;28(11):4523-30.
20. Foster DW, Bloom B. The synthesis of fatty acids by rat liver slices in tritiated water. J Biol Chem. 1963 Mar;238:888-92.
21. Sanders FWB, Griffin JL. De novo lipogenesis in the liver in health and disease: more than just a shunting yard for glucose. Biol Rev Camb Philos Soc. 2016;91(2):452-68.
22. Murphy EJ. Stable isotope methods for the in vivo measurement of lipogenesis and triglyceride metabolism. J Anim Sci. 2006;84 Suppl:E94-104.
23. Dekker MJ, Su Q, Baker C, Rutledge AC, Adeli K. Fructose: a highly lipogenic nutrient implicated in insulin resistance, hepatic steatosis, and the metabolic syndrome. Am J Physiol Endocrinol Metab. 2010;299(5):E685-94.
24. Coleman RA, Lee DP. Enzymes of triacylglycerol synthesis and their regulation. Prog Lipid Res. 2004;43(2):134-76.
25. Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005;115(5):1343-51.
26. Shimano H. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog Lipid Res. 2001;40(6):439-52.
27. Osborne TF. Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action. J Biol Chem. 2000;275(42):32379-82.
28. Horton JD, Bashmakov Y, Shimomura I, Shimano H. Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice. Proc Natl Acad Sci U S A. 1998;95(11):5987-92.
29. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest. 2002;109(9):1125-31.
30. Ferre P, Foufelle F. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab. 2010;12 Suppl 2:83-92.
31. Kohjima M, Higuchi N, Kato M, Kotoh K, Yoshimoto T, Fujino T, et al. SREBP-1c, regulated by the insulin and AMPK signaling pathways, plays a role in nonalcoholic fatty liver disease. Int J Mol Med. 2008;21(4):507-11.
32. Lima-Cabello E, García-Mediavilla M, Miquilena-Colina M, Vargas-Castrillón J, Lozano-Rodríguez T, Fernández-Bermejo M, et al. Enhanced expression of pro-inflammatory mediators and liver X-receptor-regulated lipogenic genes in non-alcoholic fatty liver disease and hepatitis C. Clin Sci (Lond). 2010; 120(6):239-50.
33. Foretz M, Pacot C, Dugail I, Lemarchand P, Guichard C, Le Liepvre X, et al. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose. Mol Cell Biol. 1999;19(5):3760-8.
34. Azzout-Marniche D, Becard D, Guichard C, Foretz M, Ferre P, Foufelle F. Insulin effects on sterol regulatory-element-binding protein-1c (SREBP-1c) transcriptional activity in rat hepatocytes. Biochem J. 2000;350 Pt 2:389-93.
35. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, et al. Role of LXRs in control of lipogenesis. Genes Dev. 2000;14(22):2831-8.
36. Brown MS, Goldstein JL. Selective versus total insulin resistance: a pathogenic paradox. Cell Metab. 2008;7(2):95-6.
37. Moore DD. Nuclear receptors reverse McGarry›s vicious cycle to insulin resistance. Cell Metab. 2012;15(5):615-22.
38. Hijmans BS, Grefhorst A, Oosterveer MH, Groen AK. Zonation of glucose and fatty acid metabolism in the liver: mechanism and metabolic consequences. Biochimie. 2014;96:121-9.
39. Jitrapakdee S. Transcription factors and coactivators controlling nutrient and hormonal regulation of hepatic gluconeogenesis. Int J Biochem Cell Biol. 2012;44(1):33-45.
40. Chavez JA, Summers SA. Lipid oversupply, selective insulin resistance, and lipotoxicity: molecular mechanisms. Biochim Biophys Acta. 2010;1801(3):252-65.
41. Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell. 2000;6(1):77-86.
42. Cook JR, Langlet F, Kido Y, Accili D. Pathogenesis of selective insulin resistance in isolated hepatocytes. J Biol Chem. 2015;290(22):13972-80.
43. Neuschwander-Tetri BA, Loomba R, Sanyal AJ, Lavine JE, Van Natta ML, Abdelmalek MF, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet (Lond). 2015;385(9972):956-65.
44. Wang Q, Jiang L, Wang J, Li S, Yu Y, You J, et al. Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice. Hepatology. 2009;49(4):1166-75.
45. Loomba R, Kayali Z, Noureddin M, Ruane P, Lawitz EJ, Bennett M, et al. GS-0976 Reduces Hepatic Steatosis and Fibrosis Markers in Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology. 2018;155(5):1463-73.e6.
46. Ishimoto T, Lanaspa MA, Le MT, Garcia GE, Diggle CP, Maclean PS, et al. Opposing effects of fructokinase C and A isoforms on fructose-induced metabolic syndrome in mice. Proc Natl Acad Sci U S A. 2012;109(11):4320-5.
47. Evans RM, Barish GD, Wang YX. PPARs and the complex journey to obesity. Nat Med. 2004;10(4):355-61.
48. Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med. 2002;53:409-35.
49. Lefebvre P, Chinetti G, Fruchart JC, Staels B. Sorting out the roles of PPAR alpha in energy metabolism and vascular homeostasis. J Clin Invest. 2006;116(3):571-80.
50. Motojima K, Passilly P, Peters JM, Gonzalez FJ, Latruffe N. Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner. J Biol Chem. 1998 ;273(27):16710-4.
51. Leone TC, Weinheimer CJ, Kelly DP. A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders. Proc Natl Acad Sci U S A. 1999;96(13):7473-8.
52. Aoyama T, Peters JM, Iritani N, Nakajima T, Furihata K, Hashimoto T, et al. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem.1998;273(10):5678-84.
53. Patel DD, Knight BL, Wiggins D, Humphreys SM, Gibbons GF. Disturbances in the normal regulation of SREBP-sensitive genes in PPAR alpha-deficient mice. J Lipid Res. 2001;42(3):328-37.
54. Miller CW, Ntambi JM. Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase 1 gene expression. Proc Natl Acad Sci U S A. 1996;93(18):9443-8.
55. Chen G, Liang G, Ou J, Goldstein JL, Brown MS. Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver. Proc Natl Acad Sci U S A. 2004;101(31):11245-50.
56. Fernandez-Alvarez A, Alvarez MS, Gonzalez R, Cucarella C, Muntane J, Casado M. Human SREBP1c expression in liver is directly regulated by peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem. 2011;286(24):21466-77.
57. Berlanga A, Guiu-Jurado E, Porras JA, Auguet T. Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol. 2014;7:221-39.
58. Nemoto Y, Saibara T, Ogawa Y, Zhang T, Xu N, Ono M, et al. Tamoxifen-induced nonalcoholic steatohepatitis in breast cancer patients treated with adjuvant tamoxifen. Intern Med. 2002;41(5):345-50.
59. Giordano Attianese GM, Desvergne B. Integrative and systemic approaches for evaluating PPARbeta/delta (PPARD) function. Nucl Recept Signal.2015;13:e001.
60. Ricote M, Glass CK. PPARs and molecular mechanisms of transrepression. Biochim Biophys Acta. 2007;1771(8):926-35.
61. Wang YX, Lee CH, Tiep S, Yu RT, Ham J, Kang H, et al. Peroxisome-proliferator-activated receptor delta activates fat metabolism to prevent obesity. Cell. 2003;113(2):159-70.
62. Ajmer SG, Meenu B, Deepti P, Bhupinder SS, Viney L. Recent Updates on Peroxisome Proliferator-Activated Receptor δ Agonists for the Treatment of Metabolic Syndrome. Med Chem. 2016;12(1):3-21.
63. Neuschwander-Tetri BA. Therapeutic Landscape for NAFLD in 2020. Gastroenterology. 2020;158(7):1984-98.e3.
64. Hudgins LC, Seidman CE, Diakun J, Hirsch J. Human fatty acid synthesis is reduced after the substitution of dietary starch for sugar. Am J Clin Nutr. 1998;67(4):631-9.
65. Parks EJ, Krauss RM, Christiansen MP, Neese RA, Hellerstein MK. Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance. J Clin Invest. 1999;104(8):1087-96.
66. Steneberg P, Sykaras AG, Backlund F, Straseviciene J, Söderström I, Edlund H. Hyperinsulinemia Enhances Hepatic Expression of the Fatty Acid Transporter Cd36 and Provokes Hepatosteatosis and Hepatic Insulin Resistance. J Biol Chem. 2015;290(31):19034-43.
67. Bai L, Jia Y, Viswakarma N, Huang J, Vluggens A, Wolins NE, et al. Transcription coactivator mediator subunit MED1 is required for the development of fatty liver in the mouse. Hepatology. 2011;53(4):1164-74.
68. Lee YS, Park JS, Lee DH, Lee D-K, Kwon SW, Lee B-W, et al. The Antidiabetic Drug Lobeglitazone Protects Mice From Lipogenesis-Induced Liver Injury via Mechanistic Target of Rapamycin Complex 1 Inhibition. Front Endocrinol. 2018;9:539.
69. Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem. 1996;271(18):10697-703.
70. Berg AH, Combs TP, Scherer PE. ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends in endocrinology and metabolism: TEM. 2002;13(2):84-9.
71. Yu JG, Javorschi S, Hevener AL, Kruszynska YT, Norman RA, Sinha M, et al. The effect of thiazolidinediones on plasma adiponectin levels in normal, obese, and type 2 diabetic subjects. Diabetes. 2002;51(10):2968-74.
72. Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 2010;362(18):1675-85.
73. Bril F, Kalavalapalli S, Clark VC, Lomonaco R, Soldevila-Pico C, Liu IC, et al. Response to Pioglitazone in Patients With Nonalcoholic Steatohepatitis With vs Without Type 2 Diabetes. Clin Gastroenterol Hepatol. 2018;16(4):558-66.e2.
74. Ratziu V, Harrison SA, Francque S, Bedossa P, Lehert P, Serfaty L, et al. Elafibranor, an Agonist of the Peroxisome Proliferator-Activated Receptor-α and -δ, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening. Gastroenterology. 2016;150(5):1147-59.e5.
75. Wolfrum C, Stoffel M. Coactivation of Foxa2 through Pgc-1beta promotes liver fatty acid oxidation and triglyceride/VLDL secretion. Cell Metab. 2006;3(2):99-110.
76. Bochkis IM, Rubins NE, White P, Furth EE, Friedman JR, Kaestner KH. Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress. Nat Med. 2008;14(8):828-36.
77. Habegger KM, Heppner KM, Geary N, Bartness TJ, DiMarchi R, Tschop MH. The metabolic actions of glucagon revisited. Nat Rev Endocrinol. 2010;6(12):689-97.
78. von Meyenn F, Porstmann T, Gasser E, Selevsek N, Schmidt A, Aebersold R, et al. Glucagon-induced acetylation of Foxa2 regulates hepatic lipid metabolism. Cell Metab. 2013 Mar 5;17(3):436-47.
79. Wolfrum C, Asilmaz E, Luca E, Friedman JM, Stoffel M. Foxa2 regulates lipid metabolism and ketogenesis in the liver during fasting and in diabetes. Nature. 2004;432(7020):1027-32.
80. Howell JJ, Stoffel M. Nuclear export-independent inhibition of Foxa2 by insulin. The J Biol Chem. 2009;284(37):24816-24.
81. Wan M, Leavens KF, Saleh D, Easton RM, Guertin DA, Peterson TR, et al. Postprandial hepatic lipid metabolism requires signaling through Akt2 independent of the transcription factors FoxA2, FoxO1, and SREBP1c. Cell Metab. 2011;14(4):516-27.
82. Chatterjee P, Seal S, Mukherjee S, Kundu R, Mukherjee S, Ray S, et al. Adipocyte fetuin-A contributes to macrophage migration into adipose tissue and polarization of macrophages. J Biol Chem. 2013;288(39):28324-30.
83. Denecke B, Gräber S, Schäfer C, Heiss A, Wöltje M, Jahnen-Dechent W. Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A. Biochem J. 2003;376(Pt 1):135-45.
84. Mori K, Emoto M, Yokoyama H, Araki T, Teramura M, Koyama H, et al. Association of serum fetuin-A with insulin resistance in type 2 diabetic and nondiabetic subjects. Diabetes Care. 2006;29(2):468.
85. Mathews ST, Singh GP, Ranalletta M, Cintron VJ, Qiang X, Goustin AS, et al. Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes. 2002;51(8):2450-8.
86. Stefan N, Hennige AM, Staiger H, Machann J, Schick F, Kröber SM, et al. Alpha2-Heremans-Schmid glycoprotein/fetuin-A is associated with insulin resistance and fat accumulation in the liver in humans. Diabetes Care. 2006;29(4):853-7.
87. Reinehr T, Roth CL. Fetuin-A and its relation to metabolic syndrome and fatty liver disease in obese children before and after weight loss. J Clin Endocrinol Metab. 2008;93(11):4479-85.
88. Dogru T, Genc H, Tapan S, Aslan F, Ercin CN, Ors F, et al. Plasma fetuin-A is associated with endothelial dysfunction and subclinical atherosclerosis in subjects with nonalcoholic fatty liver disease. Clin Endocrinol. 2013;78(5):712-7.
89. Pérez-Sotelo D, Roca-Rivada A, Larrosa-García M, Castelao C, Baamonde I, Baltar J, et al. Visceral and subcutaneous adipose tissue express and secrete functional alpha2hsglycoprotein (fetuin a) especially in obesity. Endocrine. 2017;55(2):435-46.
90. Hennige AM, Staiger H, Wicke C, Machicao F, Fritsche A, Häring HU, et al. Fetuin-A induces cytokine expression and suppresses adiponectin production. PLoS One. 2008;3(3):e1765.
91. Heinrichsdorff J, Olefsky JM. Fetuin-A: the missing link in lipid-induced inflammation. Nat Med. 2012;18(8):1182-3.
92. Nishimura T, Nakatake Y, Konishi M, Itoh N. Identification of a novel FGF, FGF-21, preferentially expressed in the liver. Biochim Biophys Acta. 2000;1492(1):203-6.
93. Fon Tacer K, Bookout AL, Ding X, Kurosu H, John GB, Wang L, et al. Research resource: Comprehensive expression atlas of the fibroblast growth factor system in adult mouse. Mol Endocrinol. 2010;24(10):2050-64.
94. Zhang X, Yeung DCY, Karpisek M, Stejskal D, Zhou ZG, Liu F, et al. Erratum. Serum FGF21 Levels Are Increased in Obesity and Are Independently Associated With the Metabolic Syndrome in Humans. Diabetes 2008;57:1246-1253. Diabetes. 2019;68(1):235.
95. Mraz M, Bartlova M, Lacinova Z, Michalsky D, Kasalicky M, Haluzikova D, et al. Serum concentrations and tissue expression of a novel endocrine regulator fibroblast growth factor-21 in patients with type 2 diabetes and obesity. Clin Endocrinol. 2009;71(3):369-75.
96. Dushay J, Chui PC, Gopalakrishnan GS, Varela-Rey M, Crawley M, Fisher FM, et al. Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease. Gastroenterology. 2010;139(2):456-63.
97. Li H, Fang Q, Gao F, Fan J, Zhou J, Wang X, et al. Fibroblast growth factor 21 levels are increased in nonalcoholic fatty liver disease patients and are correlated with hepatic triglyceride. J Hepatol. 2010;53(5):934-40.
98. Zarei M, Pizarro-Delgado J, Barroso E, Palomer X, Vázquez-Carrera M. Targeting FGF21 for the Treatment of Nonalcoholic Steatohepatitis. Trends Pharmacol Sci. 2020;41(3):199-208.
99. Jiang S, Yan C, Fang QC, Shao ML, Zhang YL, Liu Y, et al. Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis. J Biol Chem. 2014;289(43):29751-65.
100. Traussnigg S, Schattenberg JM, Demir M, Wiegand J, Geier A, Teuber G, et al. Norursodeoxycholic acid versus placebo in the treatment of non-alcoholic fatty liver disease: a double-blind, randomised, placebo-controlled, phase 2 dose-finding trial. Lancet Gastroenterol Hepatol. 2019;4(10):781-93.
101. Ambros V. The functions of animal microRNAs. Nature. 2004;431(7006):350-5.
102. Szabo G, Csak T. Role of MicroRNAs in NAFLD/NASH. D Dig Dis Sci. 2016;61(5):1314-24.
103. Cheung O, Puri P, Eicken C, Contos MJ, Mirshahi F, Maher JW, et al. Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression. Hepatology. 2008;48(6):1810-20.
104. Xu H, He JH, Xiao ZD, Zhang QQ, Chen YQ, Zhou H, et al. Liver-enriched transcription factors regulate microRNA-122 that targets CUTL1 during liver development. Hepatology. 2010;52(4):1431-42.
105. Laudadio I, Manfroid I, Achouri Y, Schmidt D, Wilson MD, Cordi S, et al. A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation. Gastroenterology. 2012;142(1):119-29.
106. Deng XG, Qiu RL, Wu YH, Li ZX, Xie P, Zhang J, et al. Overexpression of miR-122 promotes the hepatic differentiation and maturation of mouse ESCs through a miR-122/FoxA1/HNF4a-positive feedback loop. Liver Int. 2014;34(2):281-95.
107. Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 2006;3(2):87-98.
108. Griebeler EM, Werner J. Formal comment on: Myhrvold (2016) Dinosaur metabolism and the allometry of maximum growth rate. PLoS ONE; 11(11): e0163205. PLoS One. 2018;13(2):e0184756.
109. Tryndyak VP, Latendresse JR, Montgomery B, Ross SA, Beland FA, Rusyn I, et al. Plasma microRNAs are sensitive indicators of inter-strain differences in the severity of liver injury induced in mice by a choline- and folate-deficient diet. Toxicol Appl Pharmacol. 2012;262(1):52-9.
110. Povero D, Eguchi A, Li H, Johnson CD, Papouchado BG, Wree A, et al. Circulating extracellular vesicles with specific proteome and liver microRNAs are potential biomarkers for liver injury in experimental fatty liver disease. PLoS One. 2014;9(12):e113651.
111. Kakazu E, Mauer AS, Yin M, Malhi H. Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner. J Lipid Res. 2016;57(2):233-45.
112. Povero D, Eguchi A, Niesman IR, Andronikou N, de Mollerat du Jeu X, Mulya A, et al. Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells. Sci Signal. 2013;6(296):ra88.
113. Hirsova P, Ibrahim SH, Krishnan A, Verma VK, Bronk SF, Werneburg NW, et al. Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes. Gastroenterology. 2016;150(4):956-67.
114. Ibrahim SH, Hirsova P, Tomita K, Bronk SF, Werneburg NW, Harrison SA, et al. Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes. Hepatology. 2016;63(3):731-44.
115. Cannito S, Morello E, Bocca C, Foglia B, Benetti E, Novo E, et al. Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis. PLoS One. 2017;12(3):e0172575.
116. Hernández A, Arab JP. Extracellular Vesicles in NAFLD/ALD: From Pathobiology to Therapy. Cells. 2020;9(4):817.