Hepatic lipid metabolism and non-alcoholic fatty liver disease

Nutrition, Metabolism & Cardiovascular Diseases (2009) 19, 291e302

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nmcd

REVIEW

Hepatic lipid metabolism and non-alcoholic fatty liver disease* P. Tessari*, A. Coracina, A. Cosma, A. Tiengo Dept. of Clinical and Experimental Medicine, Chair of Metabolism, University of Padova, Italy Received 22 April 2008; received in revised form 10 December 2008; accepted 29 December 2008

KEYWORDS Fatty liver; Insulin; Metabolic syndrome; Hepatic lipid metabolism; Microsome; Peroxidation; Gene expression; Lipoprotein export; Metformin; Apoptosis

Abstract Non-alcoholic fatty liver disease (NAFLD) is an increasingly recognized pathology with a high prevalence and a possible evolution to its inflammatory counterpart (non-alcoholic steatohepatitis, or NASH). The pathophysiology of NAFLD and NASH has many links with the metabolic syndrome, sharing a causative factor in insulin resistance. According to a two-hit hypothesis, increased intrahepatic triglyceride accumulation (due to increased synthesis, decreased export, or both) is followed by a second step (or ‘‘hit’’), which may lead to NASH. The latter likely involves oxidative stress, cytochrome P450 activation, lipid peroxidation, increased inflammatory cytokine production, activation of hepatic stellate cells and apoptosis. However, both ‘‘hits’’ may be caused by the same factors. The aim of this article is to overview the biochemical steps of fat regulation in the liver and the alterations occurring in the pathogenesis of NAFLD and NASH. ª 2009 Elsevier B.V. All rights reserved.

Introduction Non-alcoholic fatty liver disease (NAFLD), the most common liver disease, is defined by a hepatic triglyceride content exceeding 5% of liver weight [1], although a ‘‘normal’’ triglyceride liver content in healthy, lean and

*

Disclosure Statement: The authors have nothing to disclose. * Corresponding author. Dept. of Clinical and Experimental Medicine, Chair of Metabolism, Policlinico Universitario, via Giustiniani 2, 35128 Padova, Italy. Tel.: þ39 049 8211748; fax: þ39 049 8754179. E-mail address: [email protected] (P. Tessari).

middle-aged humans with low transaminase concentrations has been recently set to 1.9% of organ weight and to 3.9% in the ‘‘general’’ population [2]. NAFLD may also have a genetic predisposition [3]. NAFLD prevalence is 10e24%, increasing to 25e75% in obesity and type II diabetes mellitus [4,5]. NAFLD is associated with insulin resistance, hypertriglyceridemia and, more generally, to the metabolic syndrome [6]. In nondiabetic subjects, there is a correlation between body mass index and liver fat [7]. Fatty liver disease is a major contributor to cardiovascular and overall obesity-related morbidity and mortality [8,9]. NAFLD accounts for z90% of cases of asymptomatic elevation of transaminases when other causes of liver

0939-4753/$ - see front matter ª 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2008.12.015

292 disease are excluded [5]. Nonetheless, transaminases are normal in >80% of subjects with NAFLD [10]. Although a ‘‘benign’’ condition, NAFLD is a risk factor for more serious liver alterations (necroinflammation, hepatocyte ballooning, Mallory bodies formation, enlargement and dysfunction of mitochondria), eventually leading to fibrosis and to non-alcoholic steatohepatitis (NASH). NASH in turn affects z3% of the general lean population, z20% of obese subjects and z50% of morbidly obese subjects [5]. About 10% of NAFLD patients eventually progress to NASH. This rate represents the net balance between 12 and 40% of NAFLD converted to NASH and 16e28% of NASH reverted to NAFLD. NASH may progress to cirrhosis and to liver-related death (mostly hepatocellular carcinoma) in z25% and z10% of cases, respectively [4]. Two steps (or ‘‘hits’’) have been proposed for the pathophysiology of NAFLD and NASH [4,11], although this theory has recently been challenged. The first ‘‘hit’’ is insulin resistance leading to NAFLD. The second is oxidative stress, determining lipid peroxidation, increased cytokine production and inflammation, ultimately resulting in NASH [12]. Dietary factors may modulate liver steatosis: a diet high in saturated fat increases liver lipids and plasma insulin levels, inducing insulin resistance [13,14], and affecting mitochondrial function. Inflammatory stimuli play a role in the progression of NAFLD to NASH through the activation of nuclear receptors. Liver cells are involved in many pathways of lipid metabolism (Table 1) and also according to their location within the lobule. The aim of this review is to provide a concise update on fatty acid and lipoprotein metabolism in the liver particularly in relationship to NAFLD and NASH, in the context of other causative factors.

Hepatic zonation of lipid metabolic activities On the basis of enzyme and metabolite distributions in selected cell cultures of either periportal (i.e. afferent) or perivenous - (i.e. efferent)-enriched hepatocyte populations, and using the isolated liver following orthograde vs. retrograde perfusion [15], two functionally-specialized zones have been proposed. The periportal zone is the site of oxidative energy metabolism, fatty acid b-oxidation, amino acid catabolism, ureagenesis, gluconeogenesis, cholesterol synthesis and degradation [16], bile formation and detoxifying metabolic pathways. Conversely, the perivenous zone is the site of glycolysis, glycogen synthesis from glucose, de novo lipid synthesis, ketogenesis, glutamine formation, and xenobiotic metabolism. These zones

Table 1 Liver metabolic activities related to lipid metabolism.      

hepatic zonation of lipid metabolism liposynthesis de novo lipogenesis fatty acid b-oxidation lipolysis lipoprotein synthesis and export

P. Tessari et al. are characterized by differences in blood flow and innervation, which determine concentration gradients of oxygen, substrates and hormones, as well as by differences in nerve density. The differential gene expression in upstream and downstream hepatocytes can also be caused by the zonal gradients of oxygen and hormone concentrations. Fatty acid oxidation occurs in the mitochondria, and in the cytosol in peroxisomes and microsomes. The distribution of palmitate oxidation in rat mitochondria within the acinar cells is flexible, and changes markedly with the physiological status [17]. The [periportal/perivenous] ratio of oxidation was 1.5, 2.0, 1.0 and 0.4 in fed, starved, refed and cold-exposed rats, respectively [17], and it was paralleled by zonation of the carnitine palmitoyltransferase-1 (CPT-1) activity (in fed and in cold-exposed animals), as well as of the mitochondrial 3-hydroxy-3-methyl-glutarylCoA synthase activity (in starved animals). In contrast, no differences as regards sensitivity of CPT-1 to malonyl-CoA, the intracellular concentration of malonyl-CoA, fatty acid synthase [FAS] activity, acetyl-CoA carboxylase activity, and the relative content of the two hepatic acetyl-CoA carboxylase isoforms, were detected [17]. However, peroxisomes palmitate oxidation was always preferentially located in the perivenous hepatic areas, irrespective of the physiological status of the animal. Thus, the changes in the acinar distribution of mitochondrial long-chain fatty acid oxidation involve specific mechanisms under different physiological conditions.

Liposynthesis and de novo lipogenesis The esterification of free fatty acids (activated as acyl-CoA) with glycerol (activated as alpha-glycerophosphate [a-GP]) (i.e. liposynthesis) is driven by the key enzymes glycerophosphate acyltransferase (GPAT). Both the nutritional status and insulin activate GPAT gene transcription and/or activity [18]. Glucagon inhibits GPAT [19]. An overflow of FFA to the liver stimulates both esterification and lipoprotein synthesis [20] The synthesis of FFA from acetyl-CoA (de novo lipogenesis) is stimulated by insulin first through glucokinase activation and increased glucose metabolism, then through activation of the transcription factor sterol regulatory element-binding protein-1c (SREBP-1c) [21,22] (Fig. 1). SREBP-1 in turn stimulates several lipogenic enzymes, such as liver pyruvate kinase (L-PK), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearyl-CoA desaturase (SCD) and the recently discovered enzyme Spot 14 (S14) [22]. L-PK is activated first to produce acetyl-CoA. In the liver, pyruvate is phosphorylated predominantly for lipogenesis. Subsequently, insulin stimulates acetyl-CoA carboxylation to malonyl-CoA through the activation of the cytosolic enzyme acetyl-CoA carboxylase (ACC), independently of FFA levels [23]. Malonyl-CoA is a key regulator of the partitioning of FFA between esterification and oxidation [23]. A high malonyl-CoA concentration, a reflection of active de novo lipogenesis, spares the FFAs from oxidation and directs them to esterification to produce triglycerides. Conversely, a low malonyl-CoA enhances CPT-1 activity favoring fatty acid transport into the mitochondria and b-oxidation. Glucagon enhances CPT-1 activity and

Lipid metabolism and the fatty liver

293 glucose

GLUT 2 hepatocyte glucose [glucokinase]

[6-phosphoglucose dehydrogenase]

GK

G-6-PDH

glucose 6-P 6-P-gluconate fructose 6-P

6-PG-DH

6-PFK

[6-phosphogluconate dehydrogenase]

ribulose-5 P

fructose-1,6-P ALDOLASE G3P [Liver pyruvate kinase]

DHAP

xylulose-5 P

PEP

L-PK pyruvate pyruvate

acetyl-CoA

OAA

pyruvate

citrate

mitochondria

citrate [Acetyl CoA carboxylase]

pyruvate

ATP -CL

[Spot S14 protein]

MALIC ENZYME

OAA acetyl-CoA malate

[Acetyl CoA carboxylase] malonyl-CoA [Fatty acid synthase] glycerol-P

palmitoyl-CoA [Stearoyl CoA desaturase]

triacylglycerol

3 Acyl-CoA GPAT

palmitoleyl-CoA

[Glycerol phosphate acyl transferase] SREBP-1c Known activators of genes

Insulin (through SREBP-1c) Insulin (through SREBP-1c), Glucose Insulin (through SREBP-1c), Glucose (through ChREBP)

Figure 1 Schematic representation of the glycolytic and lipogenic pathways in the liver, and the role of the sterol regulatory element-binding protein-1c (SREBP-1c) in the regulation of hepatic lipid metabolism (adapted and with permission from Foufelle F et al. Biochem J 2002 Sep 1;366(Pt 2):377e91). The enzymes indicated are induced at a transcriptional level by a high-carbohydrate diet. The known activators of their transcription are indicated at the bottom. Abbreviations used in this figure: ATP-CL, ATP citratelyase; DHAP, dihydroxyacetone 3-phosphate; G-6-PDH, glucose-6-phosphate dehydrogenase; GPAT, glycerol-phosphate acyltransferase; G3P, glyceraldehyde 3-phosphate; OAA, oxaloacetate; 6-PG-DH, 6-phosphogluconate dehydrogenase; PEP, phosphoenolpyruvate; P, phosphate; 6-PFK, 6-phosphofructo-1-kinase; SCD, stearoyl-CoA desaturase.

stimulates FFA oxidation and ketogenesis [24]. In a typical insulin-deficient, hyper-glucagonemic condition, such as uncontrolled type 1 diabetes, FFA oxidation and ketogenesis are enhanced, whereas de novo lipogenesis is impaired. However, when there is an exaggerated FFA afflux to the liver, such as in diabetic ketoacidosis, both pathways may be saturated, therefore resulting in combined hyperlipidemia and hyperketonemia. Subsequent elongation of the nascent long-chain FFA molecule is operated by fatty acid synthase (FAS), also stimulated

by insulin. Finally, insulin simulates the stearyl-CoA desaturase (SCD), which converts palmitoyl-CoA to palmitoleyl CoA [21,25]. The newly discovered regulatory protein Spot 14 (S14) [22] is rapidly upregulated by lipogenic stimuli, such as thyroid hormone and a high-carbohydrate diet, and stimulates cytosolic enzymes involved in lipogenesis. Although in insulin-resistant conditions SREBP-1c should not be activated, insulin surprisingly stimulates SREBP-1c transcription even in states of profound insulin resistance,

294 thus increasing de novo lipogenesis [21,25]. Another transcription factor, the carbohydrate-response element binding protein (ChREBP), mediates the carbohydratedependent stimulation of lipogenesis by activating L-PK [26]. Also PPAR-gamma is involved in the development of fatty liver as a downstream effector after SREBP-1c activation. However, de novo hepatic lipogenesis normally contributes by a minor portion to total hepatic triglyceride synthesis in humans [27]. Whether this pathway is more markedly stimulated in conditions of insulin resistance remains to be explored.

Fatty acid oxidation Fatty acid oxidation occurs in mitochondria, peroxisomes and microsomes (Fig. 2). Oxidation may proceed either from the 2nd carbon atom adjacent to the COOH as b-oxidation, or from the terminal carbon as u-oxidation. The b-oxidation occurs in the mitochondria and the peroxisomes, whereas u-oxidation occurs in the microsomes [21,28]. Carnitine palmitoyltransferase-1 (CPT-1), the ratelimiting step of mitochondrial fatty acid b-oxidation in skeletal muscle [29], is located in the outer mitochondrial membrane. CPT-1 controls the transport of long-chain acylCoA into the mitochondria, being allosterically inhibited by malonyl-CoA (see above) [29]. The AMP-activated protein kinase (AMPK, see also below), an enzyme upregulated by energy deprivation, directly phosphorylates and inactivates ACC thus decreasing malonyl-CoA formation, increasing FFA transport into the mitochondria as well as b-oxidation, thus restoring energy balance [30]. Exercise and skeletal muscle contraction activate AMPK [30,31], stimulate lipid oxidation and decrease lipid deposition in both liver [32] and skeletal muscle [33].

Figure 2 Effects of reactive oxygen species on metabolic, tissutal and inflammatory damage in the liver (from Browning JD, Horton JD. J Clin Invest 2004 Jul;114(2):147e52. Review with permission). Non-standard abbreviations used: ER, endoplasmic reticulum; AOX, acyl-CoA oxidase; MRC, mitochondrial respiratory chain; CYP, cytochrome P450; HNE, trans-4-hydroxy-2-nonenal; MDA, malondialdehyde.

P. Tessari et al.

Lipoprotein synthesis and export The triglycerides are bound to apolipoprotein B (Apo B) to build up the mature lipoprotein particle to be secreted (Fig. 3). Apo B synthesis is stimulated by elevated FFA and TG levels, as well as by the microsomal transfer protein (MTP), whereas it is inhibited by insulin [20,34]. Thus, with an elevated FFA afflux to the liver, and normal/decreased insulin concentrations or insulin resistance, an increased secretion of mature VLDL-Apo B is expected. The bulk of triglycerides incorporated into VLDL originate from intracellular storage pools rather than from de novo synthesis [35]. The balance between the FFA and insulin effects determines whether the triglycerides are combined with the Apo B VLDL particles and secreted or, alternatively, retained within the liver. With hyperinsulinemia, despite the increased FFA, triglycerides are maintained within the liver thus inducing steatosis. This key step may be relevant in the pathogenesis of NAFLD. Conversely, under conditions of (relative) insulin-deficiency and/or marked hepatic insulin resistance (such as uncontrolled insulin-deficient type 1 diabetes or a variety of insulin-resistant conditions) [36] and increased FFA availability, export of lipoproteins out of the liver can be normal or even increased. Such a condition may occur in type 2 diabetes where VLDL production can be normal or increased, and is not stimulated by de novo lipogenesis but is resistant to the suppressive effects of insulin.

Hepatic lipolysis The FFA incorporated into the triglyceride of the nascent VLDL particle is derived almost exclusively from hepatic triglyceride lipolysis [37], whose rate is 2- to 3-fold greater than that required to maintain triacylglycerol secretion

Figure 3 Role of insulin in the regulation of VLDL-Apo B synthesis and secretion in the liver. Insulin inhibits Apo B synthesis, whereas it stimulates lipogenesis through SREBP-1 activation. Under conditions of normal (or mildly impaired) hepatic insulin sensitivity, lipogenesis is enhanced but VLDL secretion is inhibited. Thus, in the liver this may translate into an intrahepatic lipid accumulation. However, under conditions of marked insulin resistance, VLDL synthesis and secretion may not be inhibited, thus resulting in increased VLDL secretion which may also be accompanied by intrahepatic lipid accumulation.

Lipid metabolism and the fatty liver

295

[38]. Therefore, most of the fatty acids released are returned to the intracellular pool to be oxidized. Cyclic AMP stimulates hepatic lipolysis. Changes in insulin, glucagon or in the content of hepatic triglyceride itself do not seem to modulate hepatic TG hydrolysis [38]. Changes in TG hydrolysis may affect not only apoB-100 secretion [39] but also hepatic TG content.

The role of AMP-kinase in metabolic regulation in the liver AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase which mediates cellular adaptation to environmental or nutritional stress factors, changes in energy metabolism and/or insulin resistance [40,41]. Activated AMPK inhibits energy-consuming biosynthetic pathways, such as fatty acid and sterol synthesis, whereas it activates ATP-producing catabolic pathways, such as fatty acid oxidation, through short-term regulation of specific enzymes and gene expression. ATP depletion through increased adenylate kinase activity or inhibition of respiration raise the intracellular AMP/ATP ratio resulting in AMPK activation [41]. Although the liver is usually resistant to hypoxia and has a stable ATP concentration under physiological conditions, liver AMPK can be activated following exercise, [41], nutrient deprivation and starvation [41e43], hypoxia & ischemia [44], oxidative/hyperosmotic stress [41] and chronic alcohol consumption [45]. Hepatic AMPK is also activated by adiponectin [46], and by the antidiabetic drugs metformin [32] and thiazolidinediones (TZDs) [47], supporting the role of AMPK as a potential target for the treatment of obesity and type 2 diabetes. The chemical compound 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) is a liver AMPK activator [48,49], through phosphorylation to 5-aminoimidazole-4carbonmide ribotide (ZMP) that, in turn, activates AMPK [49]. ZMP potentially targets all enzymes influenced by AMP. Liver, AMPK phosphorylates multiple sites acutely switching on alternative catabolic pathways (Tables 2e4). Berberine, a plant-derived compound, is a natural activator of AMPK promoting gene transcription of catabolic mediators while inhibiting the anabolic ones [50]; it also reduces liver fat. Also leptin [51] and leptin-like substances like CNTF (ciliary neutrophic factor) [52] activate AMPK.

Table 2

Table 3 Altered metabolic steps and signals at liver level in NAFLD.  Increased circulating non-esterified fatty acid (NEFA) pool (increased long-chain, decreased short- and medium-chain NEFA, phosphatidylcoline and plasmalogen;  SREBP-1c activation;  Increase of molecules interfering with insulin signalling (Rad, PC-1, leptin, TNF-alfa)  Adipose tissue inflammation and increased ceramide content  Change in cytokine pattern: increase of TNF-alpha, TGF-beta resistin, and IL-6; decrease of adiponectin, ghrelin and leptin;  Decreased Apo B synthesis and/or secretion; increase in markedly insulin-resistant conditions, T2DM, etc.

Altered hepatic lipid metabolism in the pathogenesis of NAFLD and NASH Increased FFAs from an impaired insulin-mediated suppression of lipolysis, an increased intrahepatic triglyceride synthesis and/or accumulation, possibly secondary to insulin resistance, should occur first in NAFLD [53,54] (Table 3). Increased FFAs, in turn, activate liver SREBP-1c and lipogenesis (Fig. 1). In the fatty liver, long-chain FFAs are elevated whereas short-chain FFAs, phosphatydylcoline and plasmalogen, are decreased. Liver insulin resistance is associated with molecules interfering with insulin signaling, such as Rad (i.e. Ras associated with diabetes), PC-1 (a membrane glycoprotein which interferes with tyrosine phosphorylation), leptin and TNF-alfa (which impairs IRS-1 phosphorylation and reduces Glut-4 expression) [6] (Table 3). Conversely, adipose tissue inflammation and increased ceramide content characterize subjects with high liver fat content, independently of obesity [55]. There is a characteristic cytokine pattern in liver steatosis: TNF-alpha, TGF-beta and IL-6 increase, whereas adiponectin, ghrelin and leptin decrease [55,57] (Table 3). Resistin may be either decreased [57] or increased [58] (see below).

Targets of AMPK in the liver. The symbols [\] and [Z] indicate increased or decreased activities.

Target enzyme

Phosphorylation site(s)

Change in activity

Resulting biological effect(s)

Acetyl-CoA carboxylase 1 Acetyl-CoA carboxylase 2 GlyceroPhosphate AcylTransferase HMG-CoA reductase MCD PFK-2/FBPase-2 mTOR eEF2-Kinase TSC2

Ser79 Ser218 unknown Ser872 unknown Ser22, Ser32 Thr2446 Ser398 Thr1227, Ser1345

Z Z Z Z \ minor effects Z \ \

Z lipogenesis \ fat oxidation Z triglyceride synthesis Z Cholesterol synthesis Z malonyl-CoA Unknown Z protein synthesis Z protein synthesis Z protein synthesis

Abbreviations: HMG: Hydroxymethylglutaryl Coenzyme A reductase, MCD: malonyl-CoA decarboxylase, PFK-2/FBPase-2: PFK, 6-phosphofructokinase-2, fructose-2,6-bisphosphatase, eEF2-Kinase: eukaryotic elongation factor 2, TSC2: tuberous sclerosis complex 2.

296 Table 4 Further altered metabolic steps and signals at liver level leading to NASH (in addition to those of Table 3).  Oxidative stress and increased ROS production, through cytochrome P450 2E1 (CYP2E1) activation and lipid peroxidation, further increase inflammatory cytokines, hepatic stellate cells activation, apoptosis;  Increased ROS production decrease ATP and nicotinamide dinucleotide concentrations, produce DNA and protein damage, impair membrane structure and function;  Mitochondrial dysfunction (respiratory chain deficiency, loss of mitochondrial cristae and with paracrystalline inclusions and paracrystalline inclusions, altering gene expression) increase of UCP-2;  Alternative FFA oxidation via the peroxisomes (b-oxidation) (by (PPAR-a activation) and the microsomes (u-oxidation);  KLF6 and TGF-beta-1 mRNAs up-regulation;

With hyperinsulinemia, Apo B synthesis and/or secretion should be decreased thus resulting in hepatic lipid accumulation [59]. However, with marked insulin resistance, a hyperafflux of FFA to the liver, combined with a lack of insulin effect in the inhibition of Apo B synthesis and secretion, could result in both an hepatic lipid accumulation and an exaggerated VLDL-Apo B secretion [60] (Table 3). The second step (or ‘‘hit’’), leading to NASH, involves oxidative stress, activation of the cytochrome P450 2E1 (CYP 2E1), lipid peroxidation, increased inflammatory cytokine production, activation of hepatic stellate cells, and apoptosis [61] (Table 4). Dysregulation of lysosomal metabolism and endoplasmic reticulum stress lead to apoptosis [62,63]. Stellate cell activation leads to fibrosis and collagen deposition. Although the study of the transition from NAFLD to NASH has experimental limitations and is largely limited to animal models, some typical biochemical findings related to oxidative stress have also been demonstrated in vivo in humans by biopsy [64] (Table 3). Oxidative stress (i.e. an imbalance between pro-oxidant and anti-oxidant activities) results in increased singlet oxygen superoxide radicals, hydrogen peroxide and hydroxyl radicals [21], collectively termed as ROS (or reactive oxygen species). FFA oxidation represents an active source of ROS. Increased ROS, in turn, decreases ATP and nicotinamide dinucleotide concentrations, produce DNA and protein damage, impair membrane structure and function through lipid peroxidation, and increase the release of proinflammatory cytokines [61,64e66]. Mitochondrial dysfunction, i.e. a respiratory chain deficiency, likely plays a key role in NASH (Table 4), as shown by loss of mitochondrial cristae and paracrystalline inclusions [54]. In NASH at a pre-cirrhotic-stage (when compared with HCV-related cirrhosis, primary biliary cirrhosis and healthy controls) sixteen genes were uniquely differentially expressed (twelve being underexpressed and four being overexpressed) in the liver [65]. Underexpressed genes included some maintaining mitochondrial function (such as copper/zinc superoxide dismutase, aldehyde oxidase, and catalase), as well as glucose-6-phospatase, alcohol dehydrogenase, elongation

P. Tessari et al. factor-TU, methylglutaryl coenzyme A (CoA), acyl-CoA synthetase, oxoacyl CoA thiolase, and ubiquitin. Overexpressed genes included complement-C3 and hepatocytederived fibrinogen-related protein genes. These changes possibly contributed to insulin resistance [65]. Genes involved in hepatic glucose and lipid metabolism, fatty acid transport, amino acid catabolism, insulin signaling, inflammation, coagulation, and cell adhesion, as well as genes involved in ceramide signaling and metabolism, have been associated with increased liver fat content [55,66]. Also, in NAFLD, genes related to inflammation (the macrophage marker CD68, the chemokines monocyte chemo-attractant protein-1 and macrophage inflammatory protein-1alpha, and plasminogen activator inhibitor-1), were overexpressed, whereas those of peroxisome proliferator-activated receptor-gamma and adiponectin were underexpressed [55]. Defects in oxidative phosphorylation, an impaired MRC (major respiratory chain) activity and a reduced ATP synthesis have been identified in the mitochondria of fatty livers [67]. When electron transfer across the respiratory chain is interrupted, electrons are conveyed to oxygen producing superoxide anions and hydrogen peroxide [68]. Mitochondrial b-oxidation can be either enhanced (as in insulin resistance-associated NASH) or inhibited (as in druginduced NASH). In both cases however, ROS are generated in excess by the damaged respiratory chain (Table 4). When mitochondrial oxidation is impaired, FFA accumulates in the cytosol and is alternatively oxidized via the peroxisomes (b-oxidation) and the microsomes (u-oxidation), resulting in further production of ROS [67,69,70] (Table 4). Products of microsomal u-oxidation are dicarboxylic fatty acids, which amplify damage by uncoupling oxidative phosphorylation [71]. Activation of the hepatic peroxisomes seems to be mediated by the peroxisome proliferator-activated receptor alpha (PPAR-a), a transcription factor that regulates both the microsomal (u-oxidation) and the peroxisomal (b-oxidation) pathways of lipid oxidation and ultimately increases ROS production. Peroxisomal b-oxidation may be impaired in NAFLD, enhancing u-oxidation and dicarboxylic fatty acid production [72]. Impaired oxidative phosphorylation is also reflected by the increased expression of UCP-2 in the livers of patients with NAFLD [73]. Insulin resistance and/or hyperinsulinemia predispose to oxidative stress and ROS production by stimulating microsomal lipid peroxidases and by decreasing mitochondrial b-oxidation. In NASH, the degree of steatosis correlated with serum thioredoxin level, a marker of oxidative stress [74]. In the fatty liver, ROS induce lipid peroxidation acting on polyunsaturated fatty acids (PUFAs) and producing highly reactive aldehydic derivatives (e.g. malondialdehyde, MDA, and trans-4hydroxyl-2-nonenal, HNE), which have long-term adverse effects on liver cells. Aldehydes further damage the cell by impairing nucleotide and protein synthesis and by interfering with glutathione. Peroxidation by ROS of the liver mitochondrial membraneassociated phospholipids further impairs mitochondrial function, the respiratory chain and mitochondrial genes, generating more ROS in a vicious cycle. PUFAs peroxidation may also lead to ApoB proteolysis thus impairing VLDL secretion [75]. Either apoptosis or necrosis may ensue, depending

Lipid metabolism and the fatty liver

Diet and NAFLD Both excess eating and low exercise are associated with obesity and the development of NAFLD [87]. An altered dietary macronutrient composition could also favour hepatic lipid accumulation and NAFLD even without weight changes [13]. Carbohydrate intake [88], features of the

metabolic syndrome [89], a higher intake of saturated fat and cholesterol, a lower intake of polyunsaturated fat [90], and a higher intake of total fat (at increased ratio between n  6/n  3 fatty acids) [91] have been associated with liver inflammation in NAFLD. Higher intakes of soft drinks and meat, and a lower intake of fish rich in omega-3 have been reported in NAFLD [92]. In this context, an association between high protein intake and insulin resistance and glucose intolerance has been reported [93]. Furthermore, methionine deficiency has been associated with hepatic injury in a dietary steatohepatitis model [94]. Therefore, the usual behavioural management of NAFLD includes gradual weight reduction and an increase in physical activity. Behavior therapy is based on principles Hepatic TG output 30

µmoles/g liver

25 20 15

*

10

* **

5 0 0

1

2

Metformin

3

hours

Controls

Hepatic KB output 100 80

µmoles/g liver

on the energy status of the cell. ROS and lipid peroxidation products also increase TNF-alpha, TGF-beta, and Fas ligand generation, causing cell death, inflammation and fibrosis. In experimental rodent models, increased lipid peroxidation differentiates between NASH and simple steatosis. Endotoxin administration releases tumor necrosis factoralpha (TNF-alpha) and provokes liver inflammation and hepatocyte injury in the fatty liver. This may be particularly relevant in the pathogenesis of NASH in patients with jejunoileal bypass [61]. In human liver specimens increased levels of 3-nitrotyrosine (3-NT), another index of lipid peroxidation, were also detected [54]. Inheritance of the hemochromatosis gene, C282Y, may or may not [61] promote the fibrotic progression in NASH. Expression of the cytochrome P450 (CYP 2E1), involved in microsomal b-oxidation, is increased in both humans and animal model livers of NASH [61]. Conversely, in CYP 2E1nullizygous mice, the cytochrome CYP 4A is rather induced, operating as an alternative microsomal lipid peroxidase. Since CYP 2E1 is normally suppressed by insulin, a condition of insulin resistance would result in increased CYP 2E1 activity [61]. The progression of steatosis to NASH and fibrosis also involves stellate cell activation [74], mediated by KLF6 and TGF-beta-1 mRNAs up-regulation [76]. Thiazolidinediones improve liver fibrosis by activating a corrective tissueremodeling through inhibition of matrix protein secretion by hepatic stellate cells [77]. Interestingly, inhibition of triglyceride synthesis at the level of diacylglycerol acyltransferase (DGAT)-1 with antisense oligonucleotides reduced stellate cell activation and fibrosis, suggesting a potential therapeutic target [78]. The above-outlined pathophysiological mechanisms provide some rationale for drug therapy. The antidiabetic drugs metformin [32,79], and thiazolidinediones [47,79] have been used to treat liver steatosis. In animal models of obesity and fatty liver, in vivo metformin administration reduced triglyceride output while increasing ketone-body production by the isolated perfused liver [80,81] (Fig. 4). Metformin reduced TNF-alpha, UCP-2 and SREBP-1c expression while increasing hepatocyte ATP concentrations, and activating AMPK [77,82]. Also rosiglitazone stimulated AMPK [79], whereas AICAR had protective effects on alcohol-induced fatty liver [83]. There are no definite criteria, apart from liver biopsy, to differentiate NAFLD from NASH. Factors that may be associated with NASH in patients with NAFLD are male gender, age, the degree of obesity, type 2 diabetes, high levels of alanine aminotransferase, aspartate aminotransferase and triglycerides, high IR-HOMA index, systemic hypertension, high concentrations of C-peptide [84], hyaluronic acid, type VI collagen [85], TNF-alpha, IL-8 [86], and serum acute phase proteins. Nevertheless, the validity of these assays is still to be ascertained.

297

60 40

*

*

20 0 0

1 Metformin

2

3

hours

Controls

Figure 4 Effects of in vivo metformin treatment of obese, hyperlipidemic rats, on triglyceride and ketone-body production in the isolated perfused liver (from Ref. [18], with permission). Time-pattern of triglyceride (upper panel) and total ketone-body (i.e. the sum of 3-hydroxybutyrate and acetoacetate) (lower panel) output by the isolated perfused rat liver during the 3-h of perfusion. Upper panel: Empty triangles: Triglyceride (TG) output from livers of hyperlipidemic animals treated in vivo with metformin for 7 days. Full squares: Livers of hyperlipidemic animals treated with water (Zcontrols). *p < 0.05, and **p < 0.01 vs. controls. Lower panel: Empty triangles: Total ketone-body (KB) output from livers of hyperlipidemic animals treated in vivo with metformin for 7 days. Full squares: Livers of hyperlipidemic animals treated with water (Zcontrols). * p < 0.05 vs. controls. Data are expressed as micromoles (Mean  SEM) of substrate per gram of liver.

298 and techniques modifying the patients’ eating and activity habits. It has recently been concluded that lifestyle modifications have favorable effects on NAFLD/NASH [95]. Dietary recommendations included a controlled calorie intake (between 1000 and 1200 kcal/day for overweight women, and between 1200 and 1600 kcal/day for overweight men and heavier or more active women), and a balanced nutrient composition, i.e. 50% of total calories as carbohydrates, 30% lipids (7e10% from saturated fat), and 20% proteins. Total calories may be gradually increased paralleling the increase in physical activity. The diets should induce a calorie deficit of 500e1000 kcal/day, hopefully resulting in a weight loss of 0.5e1.0 kg/week. Switching to a low-fat, standard diet may prevent the progression from NAFLD to NASH, although steatosis may not be prevented [96]. Since the metabolism of dietary fat is impaired in patients with NAFLD [97], a decreased total dietary fat should restrain postprandial lipemia. N-3 Fatty acid intake (specifically DHA and EPA) improve CHD risk by affecting metabolic variables such as blood triglycerides [98] and may be useful also in NAFLD. Beneficial effects on both insulin sensitivity and lipid markers have been found in response to low carbohydrates and highfiber intakes from fresh fruit, vegetables, legumes, and grains [99]. Reduced carbohydrate should decrease liver acetyl-CoA and, therefore, de novo lipid synthesis. A greater intake of mono-unsaturated fatty acids (MUFAs) (olive oil), in place of high-saturated fatty acid (SFA) foods (fatty meats and full-fat dairy products) would be recommended because SFAs have deleterious effects on liver function [63] and raise blood LDL concentrations [90]. Soft drinks should also be avoided or restrained [92].

Adipokines and NASH A disturbed adipocytokine secretion might promote hepatic steatosis and non-alcoholic steatohepatitis [100], by interfering with insulin sensitivity [101] and the inflammatory process [102,103].

P. Tessari et al. and exerts anorectic and thermogenic effects. Resistance to both insulin and leptin action have been associated with liver triglyceride accumulation [112]. Leptin may prevent lipid accumulation in non-adipose tissues such as the myocardium, skeletal muscle, pancreas and liver, through the modulation of hepatic b-oxidation [112,113]. Therefore, leptin should prevent lipotoxicity. Leptin infusion in mice reduced visceral fat and liver triglyceride storage, and it enhanced hepatic insulin action without affecting FFA concentration and peripheral glucose uptake [114]. This beneficial effect did not depend just on the enhanced insulin sensitivity, but also on a leptin action itself. Leptin lowered the expression of SREBP-1, thus promoting fatty acid b-oxidation and thermogenesis and down-regulating lipogenesis [113]. In patients with NAFLD, leptin is either decreased [56] or increased [112]. Serum leptin correlated indirectly with C-peptide but not with body mass index (BMI) [56,112]. Serum leptin, C-peptide and age were independent predictors of steatosis [112]. Hyperleptinemia in patients with NAFLD may overwhelm leptin-resistance in peripheral tissues [115,116]. Leptin, however, may also act as a profibrogenic cytokine in sinusoidal microenvironment [116].

Resistin Resistin is a 108 amino acid protein expressed in white adipose tissue and mononuclear cells. It is associated with insulin resistance in mice, although in humans its role is not well established [117]. It may have a stimulatory action on inflammatory processes [102]. Circulating resistin levels and mRNA expression in adipose tissue were increased in NAFLD patients [58]. Serum resistin was also correlated to the severity of histological damage in the liver, being higher in patients with NASH than with NAFLD. However, body weight excess and peripheral insulin resistance were not associated with high resistin levels. Experiments in rodents [118] showed that resistin is correlated with liver insulin resistance (i.e. with increased glucose production), but these data on humans need further confirmation.

Adiponectin

Ghrelin

Adiponectin has been associated with insulin sensitivity and has anti-inflammatory properties [100]. Serum adiponectin was decreased in NASH, independently from BMI and status of glucose metabolism, as well as in mice with fatty livers [104e108]. Intrahepatic expression of adiponectin receptors (with autocrine and/or paracrine actions) was inversely related to necro-inflammatory activity and fibrosis [109,110]. Low adiponectin serum concentrations and increased collagen IV, as well as IR-HOMA index, may be indirect diagnostic indexes to differentiate simple steatosis from early stage NASH [111]. Adiponectin, however, may not be clearly associated with the severity of the histopathologic damage.

Ghrelin, an orexigenic hormone, is secreted by the stomach and regulates glucose and energy haemostasis, and food intake [119]. Ghrelin acts on the lateral hypothalamus and theoretically inhibits proinflammatory cytokine secretion and antagonizes leptin action. Plasma ghrelin is increased before food intake and rapidly falls thereafter [119], it is positively associated with insulin sensitivity, and is reduced in obesity. Ghrelin administration in rodents increased appetite, body weight, glucose levels and body fat content. In both humans and rodents, plasma ghrelin concentration was inversely correlated with body weight and body fat, and its concentration increased following diet-induced weight loss and in malnourished states [119]. Thus, ghrelin is likely to be regulated by the nutritional state and body fat, with a feedback mechanism opposing changes in body composition, and with a potential key adaptive role during caloric restriction. In rats, sustained ghrelin administration enhanced oxidative muscle AKT activation, while it reduced

Leptin Leptin is a sensor of fat mass and a regulator of energy homeostasis. Leptin binds to the hypothalamic receptor

Lipid metabolism and the fatty liver liver AKT signaling, potentially contributing by this mechanism to the concomitant blood glucose increments [120]. Ghrelin was decreased in NAFLD in association with insulin resistance [121] and it circulates in acylated (A-Ghr) and desacylated (D-Ghr) forms. Compared with lean subjects, obese patients with the metabolic syndrome have lower total Ghr and D-Ghr, but comparable A-Ghr, resulting in a higher A/D-Ghr ratio [122]. Ghrelin induced tissuespecific changes in mitochondrial and lipid metabolism gene expression, favoring triglyceride deposition in liver while reducing fatty acid oxidation and AMP-activated protein kinase, with unchanged mitochondrial oxidative enzyme activities [123]. Ghrelin was decreased in NASH patients, independently from BMI and glucose metabolism [106]. On the whole, although ghrelin, like adiponectin, seems to have a role in the pathogenesis of NASH, its effect is still under debate, also because its correlation with the severity of the histopathology is questioned [124,125].

Conclusions Modern biochemical, molecular and gene expression studies have cast new light on the pathophysiological mechanism(s) leading to NAFLD. The primary event likely originates from insulin resistance variably affecting lipid and apolipoprotein metabolism. Fat accumulation directly damages the hepatocytes and is followed by an inflammatory response, cytokine production, oxidative stress, abnormal cellular signaling and activation of stellate cells. Transition from simple steatosis to inflammation is neither completely understood nor easily diagnosed without biopsy. Knowledge of these mechanisms is important, since epidemiological evidence supports the concept of steatosis as a component of the metabolic syndrome, and an important cardiovascular risk factor. Lifestyle changes and pharmacological treatments will hopefully revert liver fat accumulation and/or prevent necroinflammation, and improve the outcome for patients.

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Acknowledgements

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Grants: This study was supported by Research Grants of the University of Padova (years 2006e2007) and from a PhD program of ‘‘Fondazione Cassa di Risparmio di Padova e Rovigo’’, Italy.

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