Liver NF-κB and AP-1 DNA Binding in Obese Patients

obesity | VOLUME 17 NUMBER 5 | MAY 2009 973

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IntroductIonNonalcoholic fatty liver disease (NAFLD), a condition associated with type-2 diabetes mellitus and obesity, includes a wide disease spectrum ranging from triacylglycerol (TAG) accumulation in hepatocytes (steatosis) to steatosis with hepatocyte ballooning and spotty necrosis, mononuclear cell and/or polymorphonu-clear cell infiltration, and fibrosis (nonalcoholic steatohepatitis (NASH)) (1). Oxidative stress (2) and insulin resistance (IR) (1,3) are two major contributors to the pathogenesis of NAFLD and in the progression from steatosis to NASH.

A condition for the development of NAFLD is TAG reten-tion within hepatocytes (1), which may result from the com-bination of nutritional factors and derangement on hepatic lipid metabolism. The latter could be due to the expression of gene polymorphisms or, particularly, to the development of IR (3). In obesity, the occurrence of high levels of glu-cose and insulin increases hepatic fatty acid (FA) synthesis

from glucose (1,3). In addition, enhanced peripheral FA mobilization may also contribute to TAG accumulation by re- directing FAs to TAG synthesis due to IR (4,5). In agree-ment with this view, obese patients with liver steatosis or steatohepatitis show hepatic levels of palmitic acid and TAGs higher than those in control subjects (2,6). This hepatic FA overload may lead to higher rates of mitochondrial FA oxi-dation and reactive oxygen species (ROS) production, as shown in cell cultures subjected to TAG supplementation (7), thereby contributing to explain the onset of oxidative stress in the liver of obese NAFLD patients with steatosis. This nutritional-type of oxidative stress is characterized by (i) glutathione depletion and lower superoxide dismutase activity (2,8), (ii) higher lipid peroxidation, protein oxida-tion, and 3-nitrotyrosine reactivity (2,3,8), and (iii) increased Kupffer cell-dependent ROS production (9), with a conse-quent reduction in the antioxidant capacity of plasma (8).

Liver NF-κB and AP-1 DNA Binding in Obese PatientsLuis A. Videla1, Gladys Tapia1, Ramón Rodrigo1, Paulina Pettinelli1, Daniela Haim1, Catherine Santibañez1, A. Verónica Araya2, Gladys Smok3, Attila Csendes4, Luis Gutierrez4, Jorge Rojas4, Jaime Castillo4, Owen Korn4, Fernando Maluenda4, Juan C. Díaz4, Guillermo Rencoret4 and Jaime Poniachik5

Oxidative stress and insulin resistance (IR) are major contributors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and in the progression from steatosis to nonalcoholic steatohepatitis (NASH). Our aim was to assess nuclear factor-κB (NF-κB) and activating protein-1 (AP-1) activation and Toll-like receptor 4 (TLR4) expression as signaling mechanisms related to liver injury in obese NAFLD patients, and examined potential correlations among them, oxidative stress, and IR. Liver NF-κB and AP-1 (electromobility shift assay (EMSA)), TLR4 expression (western blot), ferric reducing ability of plasma (FRAP), and IR evolution (HOMA) were evaluated in 17 obese patients who underwent subtotal gastrectomy with gastro-jejunal anastomosis in Roux-en-Y and 10 nonobese subjects who underwent laparoscopic cholecystectomy (controls). Liver NF-κB and AP-1 DNA binding were markedly increased in NASH patients (n = 9; P < 0.05) compared to controls, without significant changes in NAFLD patients with steatosis (n = 8), whereas TLR4 expression was comparable between groups. Hepatic NF-κB activation was positively correlated with that of AP-1 (r = 0.79; P < 0.0001); both liver NF-κB and AP-1 DNA binding were inversely associated with FRAP (r = –0.43 and r = –0.40, respectively; P < 0.05) and directly correlated with HOMA (r = 0.66 and r = 0.62, respectively, P < 0.001). Data presented show enhanced liver activation of the proinflammatory transcription factors NF-κB and AP-1 in obese patients with NASH, parameters that are significantly associated to oxidative stress and IR.

Obesity (2009) 17, 973–979. doi:10.1038/oby.2008.601

1Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile; 2Department of Endocrinology, University of Chile Clinical Hospital, Santiago, Chile; 3Department of Pathological Anatomy, University of Chile Clinical Hospital, Santiago, Chile; 4Department of Surgery, University of Chile Clinical Hospital, Santiago, Chile; 5Department of Medicine, University of Chile Clinical Hospital, Santiago, Chile. Correspondence: Luis A. Videla ([email protected])

Received 20 May 2008; accepted 5 October 2008; published online 22 January 2009. doi:10.1038/oby.2008.601

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articlesintegrative Physiology

Consequently, a prolonged oxidative stress condition in the liver might favor hepatic n-3 long-chain polyunsaturated FA depletion and IR (6,10), in association with the redox acti-vation of multiple stress- sensitive serine–threonine kinases that alters insulin signaling (3).

Enhancement of hepatic oxidative stress occurs in NASH patients compared with steatosis (2,3). In addition, NASH patients exhibit (i) decreased catalase activity (8), (ii) increased 8-hydroxydeoxyguanosine reactivity as marker of oxidative DNA damage (11), (iii) upregulation of inducible nitric oxide synthase (12) and cytochrome P450 2E1 (8), and (iv) a further major increment in lipid peroxidation and ROS production by Kupffer cells (9). These changes are paralleled by signifi-cant serum markers accounting for oxidative stress (1–3,8). Thus, liver oxidative stress in NASH involves a strong and sus-tained ROS production, in which antioxidant response is not enough to reset the original level of redox homeostasis. Under these conditions, persistent changes in signal transduction

and gene expression may occur, which might promote hepa-tocellular damage by inducing severe oxidative alterations of biomolecules with loss of their functions and cell viabil-ity impairment, and activation of redox-sensitive transcrip-tion factors, with consequent upregulation of the expression of proinflammatory mediators at the Kupffer cell level (3). In view of these findings, we examined the DNA binding of nuclear factor-κB (NF-κB) and activating protein-1 (AP-1) in liver samples from control subjects and obese patients with simple steatosis and NASH, to determine whether activa-tion of these redox-sensitive transcription factors is involved in NAFLD. To assess the role of oxidative stress and IR, corre-lations between hepatic NF-κB and AP-1 activation with the total antioxidant capacity of plasma or with the homeostatic model assessment of IR (HOMA) (13) were established. In addition, hepatic expression of Toll-like receptor 4 (TLR4), a key signaling molecule promoting NF-κB and AP-1 activation (14), was also studied.

table 1 clinical and biochemical parameters in control subjects and in patients with nonalcoholic fatty liver disease as a function of the morphological characteristics of the liver

Parameter (normal range) Controls (n = 10) Steatosis (n = 8) Steatohepatitis (n = 9)

Age (years) 36 ± 4 36 ± 2 43 ± 4

Female/male ratio 10/0 6/2 5/4

Body weight (kg) 64 ± 2 120 ± 9a 115 ± 5a

BMI (<25 kg/m2) 25 ± 0.6 45 ± 3a 43 ± 1a

Waist circumference (cm) 87 ± 4 122 ± 7a 124 ± 3a

Glucose (<100 mg/dl) 87 ± 3 92 ± 4 103 ± 7

Insulin (<20 mU/ml) 9.1 ± 1.2 17.9 ± 1.7a,c 32.4 ± 3.4a,b

HOMA (<2.5) 2.0 ± 0.3 4.1 ± 0.4a,c 8.4 ± 1.1a,b

Liver parameters

Alanine aminotransferase (9–52 IU/l) 45 ± 3 44 ± 9 56 ± 9

Aspartate aminotransferase (14–36 IU/l) 26 ± 2 33 ± 9 33 ± 4

Alkaline phosphatase (38–126 IU/l) 101 ± 7 90 ± 10 112 ± 6

γ-Glutamyl transpeptidase (12–43 IU/l) 36 ± 4 33 ± 7 41 ± 5

Total bilirubin (0.2–1.3 mg/dl) 0.55 ± 0.13 0.36 ± 0.06 0.39 ± 0.04

Prothrombin time (13–20 s) 12.5 ± 0.2 12.2 ± 0.3 13.0 ± 0.2

Lipid profile

Total triglyceride (<150 mg/dl) 202 ± 41 141 ± 16 150 ± 19

Total cholesterol (<200 mg/dl) 177 ± 9 170 ± 6 199 ± 14

LDL-cholesterol (<140 mg/dl) 99 ± 6 94 ± 7 129 ± 11a,b

HDL-cholesterol (>40 mg/dl) 46 ± 3 49 ± 7 41 ± 3

Iron metabolism

Total iron (37–145 µg/dl) 72 ± 7 69 ± 7 71 ± 7

Ferritin (ng/ml) 71 ± 14 75 ± 25 126 ± 50

Transferrin (200–300 mg/dl) 251 ± 9 310 ± 16a 246 ± 12b

Transferrin saturation (20–55%) 23 ± 2 19 ± 2 25 ± 3

Normalized FRAP (µmol Fe2+/mg uric acid) 7.8 ± 0.4 6.4 ± 0.4a 5.9 ± 0.3a

Values represent means ± s.e.m. for the number of subjects indicated.FRAP, ferric reducing ability of plasma; HDL, high-density lipoprotein; HOMA, homeostasis model assessment of insulin resistance; LDL, low-density lipoprotein.The significant differences between mean values (P < 0.05), assessed by one-way ANOVA and Bonferroni’s multiple comparison test. aP < 0.05 vs. controls, bP < 0.05 vs. steatosis, cP < 0.05 vs. steatohepatitis.



Methods And ProceduresPatients and laboratory studiesSeventeen patients with NAFLD, who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in Roux-en-Y as therapy for obes-ity, were studied. Ten nonobese patients, who underwent laparoscopic cholecystectomy, were included as the control group. The protocol was explained in detail to the subjects, who gave their written informed con-sent to participate in the study before any procedure was undertaken. The Ethics Committees of the University of Chile Clinical Hospital and of the Faculty of Medicine, University of Chile approved the study pro-tocol, according to Helsinki criteria. Nutritional and alcohol consump-tion information and anthropometric measurements were obtained. Laboratory tests included serum liver parameters (alanine aminotrans-ferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, total bilirubin, and prothrombin time), metabolic param-eters (glucose, insulin, HOMA (13), total triglyceride, total cholesterol, and low-density lipoprotein and high-density lipoprotein cholesterol), and iron metabolism parameters (total iron, ferritin, transferrin, and transferrin saturation). Total antioxidant capacity of plasma was assessed through the ferric reducing ability of plasma (FRAP) (15), and results were normalized by the respective uric acid levels and expressed as µmol Fe2+/mg uric acid. Exclusion criteria included positive hepatitis B or C serology, positive antibodies (antinuclear, antimitochondrial, and antis-mooth muscle antibodies), smoking habits or nonsmokers <1-year ces-sation, and consumption of >40 g of ethanol per week.

Both control and obese patients were given a diet of 25 kcal/kg body weight (where 1 kcal = 4.184 kJ), with 30% of the energy given as lipids (10% saturated FAs, 10% monounsaturated FAs, and 10% polyunsatu-rated FAs), 15% as proteins, and 55% as carbohydrates for at least 2 days before surgery. Liver samples of ~2 cm3 for histological diagnoses and NF-κB, AP-1, and TLR4 determinations were taken during surgery. The samples were fixed in 10% formalin and paraffin embedded, and sections were stained with hematoxylin–eosin and Van Giesson’s stain. Sections of each liver sample were observed in a blinded manner and histological analysis was performed by means of previously defined protocol (16). Liver samples for biochemical determinations were frozen at −80 °C.

nF-κB and AP-1 electromobility shift assayNuclear protein extracts from liver samples were prepared according to Deryckere and Gannon (17). The samples were subjected to electromo-bility shift assay (EMSA) for assessment of NF-κB and AP-1 DNA bind-ing using the NF-κB probe 5′-AGT TGA GGG GAC TTT CCC AGG C-3′ or the AP-1 probe 5′-CGC TTG ATG AGT CAG CCG GAA-3′ (Invitrogen Life Technologies, Carlsbad, CA), labeled with α-32P-dCTP using the Klenow DNA Polymerase Fragment I (Invitrogen, Carlsbad, CA). The specificity of the reaction was determined by a competition assay using 100-fold molar excess of unlabeled DNA probes. To assess subunit composition of DNA-binding proteins, specific antibodies were used for supershift assay, namely, goat polyclonal immunoglobulin-G raised against NF-κB p50 (SC-1190X), NF-κB p65 (SC-109X), or AP-1 c-Jun (c-Jun (N)X) (Santa Cruz Biotechnology, Santa Cruz, CA). Samples were loaded on nondenaturating 6% polyacrylamide gels and run until the free probe reached the end of the gel; NF-κB and AP-1 bands were detected by autoradiography and quantified by densitom-etry using Scion Image (Scion, Frederick, MD).

Western blot analysis of tLr4Liver samples (100 mg) frozen in liquid nitrogen were homogenized and suspended in a buffer solution pH 7.9 containing 10 mmol/l 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid, 1 mmol/l EDTA, 0.6% Nonidet P-40, 150 mmol/l NaCl, and protease inhibitors (1 mmol/l phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mmol/l orthovanadate). Soluble protein fractions (50 µg) were separated on 10% polyacrylamide gels using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (18) and transferred to nitrocellu-lose membranes (19), which were blocked for 1 h at room temperature with Tris buffer saline-containing 5% nonfat dry milk. The blots were

washed with Tris buffer saline-containing 0.1% Tween 20 and hybrid-ized with rabbit polyclonal antibodies for human TLR4 (Dako, Carpinteria, CA). Mouse monoclonal antibody for rat β-actin (ICN Biomedicals, Aurora, OH) was used as internal control. After extensive washing, the antigen–antibody complexes were detected using horse-radish peroxidase labeled goat anti-rabbit IgG or goat anti-mouse IgG and a SuperSignal West Pico Chemiluminescence kit detection system (Pierce, Rockford, IL).

statistical analysesResults are expressed as means ± s.e.m. for the number of patients studied. Statistical analysis of the differences between mean values was assessed by one-way ANOVA followed by Bonferroni’s multiple com-parison test. The differences were considered statistically significant at P < 0.05. To analyze the association between different variables, the Spearman rank order correlation coefficient was used. All statistical analyses were computed using GraphPad Prism version 2.0 (GraphPad Software, San Diego, CA).

resuLtsclinical, biochemical, and histological characteristics of patientsGeneral characteristics of the studied patients are shown in Table 1. Subjects were predominantly female and exhibited comparable ages. They were divided into three groups accord-ing to the features of their hepatic histology: (i) normal liver histology (controls), (ii) steatosis (presence of macrovesicu-lar steatosis alone), and (iii) NASH (presence of steatosis and

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Figure 1 Liver nuclear factor-κB (NF-κB) DNA binding on electromobility shift assay in control subjects and in nonalcoholic fatty liver disease patients with steatosis or steatohepatitis. (a) Autoradiograph representing lanes loaded with 8-µg nuclear protein from patient 1 (control), patient 2 (steatosis), patient 3 (steatohepatitis), from steatohepatitis patient 4 in competition experiments without (positive control (pc)) and with 100-fold molar excess of the unlabeled DNA probe (com), and from steatohepatitis patient 5 in supershift analysis. (b) Bar graphs corresponding to densitometric quantification of relative NF-κB DNA binding represent means ± s.e.m. for the number of patients indicated in parentheses. Significance studies, aP < 0.05 vs. controls.



lobular inflammation with hepatocyte ballooning). Patients with steatosis or NASH were significantly more obese than con-trols, as shown by their body weight, BMI, and waist circumfer-ence being 84, 76, and 42% higher than controls, respectively. Fasting blood glucose levels were comparable among the stud-ied groups, whereas fasting insulin levels in steatosis and NASH patients were 96% and 256% higher than controls (P < 0.05), respectively, being those in NASH 81% higher than steatosis (P < 0.05). Accordingly, increased IR is established in the stea-tosis and NASH groups as determined by the 105 and 320% (P < 0.05) enhancement in the HOMA index over control values, being that in NASH 105% higher than steatosis (P < 0.05). The majority of patients were asymptomatic, with normal or mild alterations of liver functions tests, lipid profile, and in param-eters related to iron metabolism. The normalized FRAP index was 18% and 24% lower in patients with steatosis and NASH compared with control values, respectively (P < 0.05).

Liver nF-κB and AP-1 dnA bindingLiver DNA binding of NF-κB (Figure 1a) and AP-1 (Figure 2b) in control subjects was characterized by low intensity bands in the EMSA, with an image density range between 8 and 51 arbitrary units for NF-κB and between 3 and 49 arbitrary units for AP-1, in agreement with the low activation of the transcription factors observed under normal conditions (20).

Liver NF-κB (Figure 1b) and AP-1 (Figure 2b) DNA bind-ing in obese patients with steatosis was comparable to that in control patients, whereas it was enhanced by 369 and 505% in NASH patients, respectively (P < 0.05). Suppression of the EMSA NF-κB (Figure 1a) and AP-1 (Figure 2a) bands by 100- molar excess of the respective unlabeled DNA probes con-firmed the specificity of the determinations. Values of hepatic NF-κB and AP-1 DNA binding obtained from controls and NAFLD patients with steatosis or steatohepatitis were signifi-cantly correlated (r = 0.79; P < 0.0001) (Figure 3). Furthermore, the associations between NF-κB and AP-1 activation with oxi-dative stress and IR were evaluated (Figure 4). In the studied groups, liver NF-κB (r = −0.43; P < 0.05) (Figure 4a) and AP-1 (r = −0.40; P < 0.05) (Figure 4b) DNA binding negatively cor-related with normalized FRAP values as a measure of systemic oxidative stress status. Positive correlations were observed

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Figure 2 Liver activating protein-1 (AP-1) DNA binding on electromobility shift assay in control subjects and in nonalcoholic fatty liver disease patients with steatosis or steatohepatitis. (a) Autoradiograph representing lanes loaded with 8-µg nuclear protein from patient 1 (control), patient 2 (steatosis), patient 3 (steatohepatitis), from steatohepatitis patient 4 in competition experiments without (positive control (pc)) and with 100-fold molar excess of the unlabeled DNA probe (com), and from steatohepatitis patient 5 in supershift analysis. (b) Bar graphs corresponding to densitometric quantification of relative AP-1 DNA binding represent means ± s.e.m. for the number of patients indicated in parentheses. Significance studies, aP < 0.05 vs. controls.

Controls

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r = 0.79P < 0.0001

150 200 250

Figure 3 Correlation between liver nuclear factor-κB (NF-κB) and activating protein-1 (AP-1) DNA binding on electromobility shift assay in control subjects and in nonalcoholic fatty liver disease patients with steatosis or steatohepatitis.

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Figure 4 Correlations between liver (a) nuclear factor-κB (NF-κB) and (b) activating protein-1 (AP-1) DNA binding with the ferric reducing ability of plasma (FRAP) and between (c) NF-κB and (d) AP-1 DNA binding with homeostasis model assessment of insulin resistance (HOMA) in control subjects and in nonalcoholic fatty liver disease patients with steatosis or steatohepatitis.



between hepatic DNA binding of NF-κB (r = 0.66; P < 0.001) (Figure 4c) and AP-1 (r = 0.62; P < 0.001) (Figure 4d) and the HOMA index.

Liver tLr4 expressionLiver TLR4 expression was evaluated via western blotting, as shown by the blots of TLR4 and β-actin protein expression of representative patients of the studied groups (Figure 5a). The respective densitometric quantification of blots expressed as TLR4/β-actin ratios revealed no significant differences between controls and obese NAFLD patients with steatosis or NASH (Figure 5b). Liver TLR4 expression was not significantly cor-related with either NF-κB activation (r = 0.35; P = 0.16), AP-1 activation (r = 0.44; P = 0.07), normalized FRAP (r = –0.15; P = 0.57), or HOMA index (r = 0.33; P = 0.19).

dIscussIonObese NASH patients in this study showed significant enhancement in the DNA binding capacity of hepatic NF-κB and AP-1, as shown by EMSA measurements in nuclear frac-tions where they coordinate the transcriptional activation of several target genes, parameters which are not significantly modified in patients with simple steatosis, in relation to con-trols. Data about NF-κB activation are in agreement with those of the immunohistochemical measurement of NF-κB p65 subunit by means of a monoclonal antibody binding, which occurs only after inhibitor of κB dissociation (21). Liver NF-κB and AP-1 activation in NASH patients is likely to be induced by hepatic oxidative stress, as evaluated by the diminution in the systemic FRAP index. The latter change is observed

concomitantly with derangement in major liver antioxidant mechanisms and induction of cytochrome P450 2E1 with pro-oxidant behavior (8), which together with the occur-rence of substantial IR may constitute determinant factors for steatohepatitis development. The significant association found between NF-κB and AP-1 activation with the oxidative stress status is consistent with the increase in ROS production by Kupffer cells, reaching to 2.0- and 20-fold in steatosis and NASH patients, respectively (9). Activated Kupffer cells exhibit a highly pro-oxidant state that is largely due to nicotinamide-adenine dinucleotide phosphate oxidase activity, which may trigger the redox activation of NF-κB and AP-1 and the consequent expression of proinflammatory mediators (2,3). Although NF-κB and AP-1 differ in the way of regulation by the redox status (2,3), obesity represents a long-term condi-tion involving a chronic and progressive enhancement in liver oxidative stress that may overcome the feedback mechanisms responsible for NF-κB and AP-1 deactivation (22,23).

The activation of NF-κB and AP-1 can occur through dif-ferent signaling mechanisms. In the case of NF-κB, it is well established that ROS-induced activation pathways rely mainly on the inhibitor of κB-kinase complex activation, a process that may also be triggered by the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β as well as by lipopolysaccharide (14,24), through different signaling cascades (22). AP-1 activation requires de novo synthesis of the c-Jun and c-Fos proteins, followed by phosphorylation of the c-Jun component by c-Jun N-terminal kinase (JNK) for full transactivation of target genes (23). Sustained JNK activa-tion, in turn, depends on ROS production indirectly, as ROS achieve the oxidation and inhibition of JNK inactivating phos-phatases, mainly those of the mitogen-activated protein kinase phosphatase group, through a mechanism involving TNF- α-induced ROS accumulation at the mitochondrial level (22,23). Furthermore, the significant associations between liver NF-κB and AP-1 activation with oxidative stress are also observed between the latter transcription factors and IR. These findings support the contention that oxidative stress and IR are interdependent factors in obese patients with NAFLD, which might arise from a pathophysiological sequence of reinforc-ing mechanisms (3). In fact, the available evidence supports the view that the initial FA overloading of the liver in obesity (4–6) can increase mitochondrial ROS production (7) leading to (i) depletion of n-3 long-chain polyunsaturated FA, which directs FAs away from oxidation and toward TAG storage thus promoting hepatic steatosis; and (ii) the onset of IR through ROS- (25) and FA- (26) induced activation of serine– threonine kinases (JNK, inhibitor of κB-kinase, protein kinase C) that upon phosphorylation of the insulin receptor and insulin receptor substrate proteins may decrease insulin signaling (27). The progression of steatosis to NASH is associated with exac-erbation of (i) hepatic oxidative stress due to IR-dependent CYP2E1 upregulation (8), mitochondrial dysfunction, and Kupffer cell (9) or infiltrating leukocyte nicotinamide- adenine dinucleotide phosphate oxidase activation; and (ii) IR due to reinforcement of serine–threonine phosphorylation of

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Figure 5 Liver Toll-like receptor 4 (TLR4) expression evaluated via western blotting in control subjects and in nonalcoholic fatty liver disease patients with steatosis or steatohepatitis. (a) Representative blots of TLR4 and β-actin protein expression are shown for patient 1 (control), patient 2 (steatosis), and patient 3 (steatohepatitis), using 50 µg of soluble protein. (b) Bar graphs correspond to the respective densitometric quantification expressed as TLR4/β-actin ratios to compare lane-to-lane equivalency in total protein content. Values shown are expressed as means ± s.e.m. for the number of patients shown in parentheses. Statistical comparisons between all groups, P > 0.05.



components of the insulin cascade (3,27), which might be trig-gered by upregulation of TNF-α and interleukin-1β through the redox activation of NF-κB and AP-1 under conditions of excessive and prolonged oxidative stress.

In addition to these mechanisms, liver NF-κB and AP-1 activation in obese patients with NASH could be achieved via TLR4 signaling pathways, which show low activation under normal conditions, being efficiently triggered upon interaction with lipopolysaccharide (14). The recognition of lipopolysac-charide by TLR4 activates inflammatory cascades in the liver involving recruitment of several adapter molecules and activa-tion of transforming growth factor β activated kinase 1, which results in inhibitor of κB-kinase and JNK phosphorylation and NF-κB and AP-1 activation, respectively (14,24). Although data presented reveal that liver TLR4 protein expression was similar in control, steatosis and NASH patients, TLR4 signal-ing is mainly dependent on endotoxemia (14). However, serum lipopolysaccharide levels in NASH patients are controversial, being unaltered in one study (28) and increased in two reports (29,30), a discrepancy that could be related to the different methodologies used to determine endotoxemia. Thus, liver NF-κB and AP-1 activation through TLR4 signaling might be of importance in NASH patients, providing that significant endotoxemia is present and liver oxidative stress is fully devel-oped. This hypothesis is based on the finding that proximal events in TLR4 signaling are known to be ROS dependent (31), raising the possibility that ROS can contribute to NF-κB and AP-1 DNA binding by activation of the TLR4 cascade. These findings suggest that regulation of TLR4 signaling in Kupffer cells may play a pathophysiological role in NASH by contrib-uting to NF-κB and AP-1 activation, although further studies related to the expression of adaptor proteins recruited for TLR4 signaling and that of genes regulated by TLR4 are needed to verify this proposal.

The significant enhancement in liver NF-κB and AP-1 DNA binding in obese NASH patients is in agreement with Kupffer cell activation and upregulation of proinflammatory cytokine signaling. Upregulation of proinflammatory cytokine signaling is supported by the increased serum levels of TNF-α (28,32–34), interleukin-6 and interleukin-8 (32), and by the overexpression of hepatic TNF-α mRNA (35) and TNF-α receptor I mRNA (21,35). In addition, the role of adipose tissue-derived adi-pokines should also be considered. This view is supported by the significant diminution in the serum levels of adiponectin observed in NASH (33). Given the significant proinflammatory potential of NF-κB and AP-1 upregulation, these transcription factors and the signaling mechanisms involved in their activa-tion become attractive molecular targets for NASH prevention and therapy. In the setting of obesity-induced NASH, the use of inhibitors of NF-κB and AP-1 might represent a therapeutic alternative alone or, more likely, as adjuvants along with other strategies such as antioxidants and/or n-3 long-chain polyun-saturated FA supplementation (2,3,36). Accordingly, curcumin alleviates the severity of hepatic inflammation in methionine/choline diet-induced steatohepatitis in mice (37) and prevents CCl4-induced liver damage in rats (38), acting both as an

NF-κB inhibitor and as an antioxidant thus hindering proin-flammatory cytokine production (36).

In conclusion, obese patients with NASH showed substan-tial increases in liver DNA binding of NF-κB and AP-1, thus contributing to the major role of oxidative stress and IR in the pathophysiological mechanism of NASH. From these data it could be suggested that these transcriptional factors could be targets of future therapeutics interventions, such as the use of specific inhibitors of NF-κB and/or AP-1.

AcknoWLedgMentsThis work was supported by grant 1060105 (to L.A.V.) from FONDECYT (Chile). The valuable nursing assistance of José Ibarra is acknowledged.

dIscLosureThe authors declared no conflict of interest.

© 2009 The Obesity Society

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Liver NF-κB and AP-1 DNA Binding in Obese Patients