Brief Article Open Access
Copyright ©2010 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Dec 14, 2010; 16(46): 5830-5837
Published online Dec 14, 2010. doi: 10.3748/wjg.v16.i46.5830
Pro12Ala polymorphism of the peroxisome proliferator-activated receptor γ2 in patients with fatty liver diseases
Johannes W Rey, Andrea Noetel, Aline Hardt, Hans Peter Dienes, Uta Drebber, Margarete Odenthal, Institute for Pathology, University Hospital of Cologne, 50924 Cologne, Germany
Ali Canbay, Department of Gastroenterology and Hepatology, University Hospital of Essen, 45147 Essen, Germany
Hakan Alakus, Department of General, Visceral and Cancer Surgery, University Hospital of Cologne, 50924 Cologne, Germany
Axel zur Hausen, Institute for Pathology, University Hospital of Freiburg, 79104 Freiburg/Brisgau, Germany
Author contributions: Rey JW and Noetel A are equally contributed to the work; Rey JW and Noetel A performed the laboratory research and analyzed the data; Canbay A contributed to final review of the manuscript and critical discussion; zur Hausen A performed DNA isolation of the blood samples; Alakus H helped with the statistical analysis; Hardt A, Drebber U and Dienes HP were responsible for scoring; Odenthal M designed the study; Odenthal M and Noetel A wrote the manuscript.
Supported by A grant of Marga and Walter Boll foundation
Correspondence to: Margarete Odenthal, PhD, Institute for Pathology, University Hospital Cologne, Kerpener Str. 62, 50924 Cologne, Germany. m.odenthal@uni-koeln.de
Telephone: +49-221-4786367 Fax: +49-221-4786360
Received: June 18, 2010
Revised: July 27, 2010
Accepted: August 3, 2010
Published online: December 14, 2010

Abstract

AIM: To test the occurrence of the Pro12Ala mutation of the peroxisome proliferator-activated receptor-γ (PPARγ)2-gene in patients with non-alcoholic fatty liver disease (NAFLD) or alcoholic fatty liver disease (AFLD).

METHODS: DNA from a total of 622 specimens including 259 blood samples of healthy blood donors and 363 histologically categorized liver biopsies of patients with NAFLD (n = 263) and AFLD (n = 100) were analyzed by Real-time polymerase chain reaction using allele-specific probes.

RESULTS: In the NAFLD and the AFLD collective, 3% of the patients showed homozygous occurrence of the Ala12 PPARγ2-allele, differing from only 1.5% cases in the healthy population. In NAFLD patients, a high incidence of the Ala12 mutant was not associated with the progression of fatty liver disease. However, we observed a significantly higher risk (odds ratio = 2.50, CI: 1.05-5.90, P = 0.028) in AFLD patients carrying the mutated Ala12 allele to develop inflammatory alterations. The linkage of the malfunctioning Ala12-positive PPARγ2 isoform to an increased risk in patients with AFLD to develop severe steatohepatitis and fibrosis indicates a more prominent anti-inflammatory impact of PPARγ2 in progression of AFLD than of NAFLD.

CONCLUSION: In AFLD patients, the Pro12Ala single nuclear polymorphism should be studied more extensively in order to serve as a novel candidate in biomarker screening for improved prognosis.

Key Words: Single nucleotide polymorphism; Peroxisome proliferator-activated receptor γ; Non-alcoholic steatohepatitis; Alcoholic steatohepatitis; Inflammation; Fibrosis; Hepatitis; Steatosis; Steatohepatitis



INTRODUCTION

Fatty liver diseases are becoming a common cause of chronic liver diseases in the Western countries encountering in about 20% of the general adult and child population[1-5]. Excessive accumulation of triglycerides in hepatocytes occurring in etiologically diverse conditions causes hepatic steatosis characterized by more than 5%-10% fat stored either in macrovesicles or in microvesicles of hepatocytes[6,7]. Whereas in the past, regular and excess alcohol consumption was the most common reason for hepatic steatosis[8,9], fatty liver diseases are now most frequently associated with obesity, insulin resistance and type 2 diabetes due to an unbalanced and rich diet in industrial nations[3,5].

The spectrum of fatty liver diseases (FLD) independent of causative agents ranges from simple steatosis to steatohepatitis, which can progress to liver fibrosis ending up in cirrhosis or hepatocellular carcinoma[3,5,10-13]. Thus, 5%-10% of non-alcoholic fatty liver disease (NAFLD) patients with steatosis develop a steatohepatitis accompanied by a high risk of progression to fibrosis[3,4]. Although fatty liver diseases can have various causes, features of steatohepatitis in NAFLD and alcoholic fatty liver disease (AFLD) are difficult to distinguish histologically[6,7]. Both are characterized by foci of liver cell necrosis and lobular inflammatory infiltrates with polymorphonuclear leukocytes. Furthermore, the onset of steatohepatitis is accompanied by ballooned hepatocytes, often harboring Mallory’s hyaline and megamitochondria or undergoing apoptosis[14]. Whereas steatosis seems to be more pronounced in non-alcoholic steatohepatitis (NASH) than in alcoholic steatohepatitis (ASH), features of necroinflammatory and cholestatic activity are more prominent in ASH liver biopsies[6]. Progression of steatohepatitis then results in pericellular fibrosis[6,7,15] involving myofibroblastic activation of sinusoidal hepatic stellate cells responsible for elevated extracellular matrix deposition[16].

Members of the peroxisome proliferator-activated receptors (PPAR) seem to play a key role in the pathophysiology of FLD by modulating increased glucose uptake and hepatic triglyceride accumulation, but also perform anti-inflammatory signals when steatohepatitis has occurred[17-19]. The PPAR family consists of PPARα, PPARγ, and PPARδ nuclear receptors, functioning as transcription factors, that mediate transcriptional response to insulin resulting in glucose uptake, increased fatty acid oxidation, lipogenesis and lipid storage, respectively[17]. Whereas the PPARα is highly present in hepatocytes, the splice variants PPARγ1 and 2 triggering adipogenesis are mainly expressed in adipose tissues and only to a minor extent in the liver. PPARγ increases the expression of genes that promote fatty acid storage, whereas it represses genes that induce lipolysis in adipocytes. In patients suffering from FLD, hepatic expression of PPARγ is shown to be involved in insulin sensitivity, triglyceride clearance and hepatic steatosis[20].

Due to its high impact as an insulin-sensitising transcription factor involved in adipogenesis and lipogenesis, the occurrence of single nucleotide polymorphisms (SNP) in the PPARγ gene was recently addressed by numerous reports studying subjects with insulin resistance, type 2 diabetes, arteriosclerosis, and hypertension[21-23]. A prevalent SNP association with impaired lipid homeostasis was observed in terms of the N-terminal proline alanine exchange (Pro12Ala) of the extra domain in the PPARγ2 variant. This PPARγ splice form includes 30 additional amino acids[24], which are responsible for a 5-6-fold increase of PPARγ’s transcriptional activity. The Pro12Ala exchange in the activating extra region of the PPARγ2 is the result of a cytosine to guanine substitution in the PPARγ gene, as a consequence encoding the Ala-allele form with a heavily reduced function[23]. In several populations, the association of the Pro12Ala polymorphism with insulin-sensitivity, type 2 diabetes, obesity and adipositas have been shown[25-27]. However, the role of the Pro12Ala polymorphism of PPARγ gene in occurrence and progression of fatty liver diseases is not yet defined.

In the present study, we analyzed the frequency of the Pro12Ala polymorphism in the PPARγ gene by a highly sensitive LNA-probe based polymerase chain reaction (PCR) approach in a total of 622 subjects of a Caucasian population, suffering from fatty liver disease (n = 359) or being healthy blood donors (n = 263). In agreement with reports showing a high Ala allele prevalence in patients with impaired lipid metabolism in obese and adipose patients[26,28], in FLD patients the Ala allele also occurs more often than in the healthy control group. Interestingly, the interpretation and linkage of the allele frequency to histological evaluation and clinical data demonstrates a prominent risk in AFLD patients bearing the Ala allele to develop severe steatohepatitis and fibrosis. Furthermore, our data revealed for the first time a higher anti-inflammatory impact of PPARγ in progression of human AFLD than NAFLD.

MATERIALS AND METHODS
Patients, biopsies and liver disease classification

From a total of 622 cases, 259 blood samples and 363 biopsies were studied for occurrence of the Pro12Ala exchange in the PPARγ gene. Local research ethics guidelines were followed. We collected 363 cases from the files of the Department of Gastroenterology and Hepatology, University Hospital of Essen (GER) and the Institute for Pathology, University Hospital of Cologne (GER) according to their histological criteria of fatty liver disease (Table 1). 263 tissue specimens from patients were classified as NAFLD according to the clinical information about alcohol consumption (less than 20 g alcohol per day). One hundred specimens of patients who consume more than 20 g alcohol per day met the definition of AFLD as described by Neuschwander-Tetri et al[29]. Clinical data, such as GOT, GPT, and γGT, were compiled along with the state of diabetes. There was no appreciable difference between the mean age of AFLD (53.93 ± 10.63, range 20-81 years) and NAFLD (50.48 ± 15.25, range 16-80 years). All specimens, stained with haematoxylin and eosin (HE) and by the Gomori method for visualization of reticular fibers, were independently classified by three experienced liver pathologists (Hardt A, Drebber U, Dienes HP), according to the histological score described by Kleiner et al[15] (Table 1). Additionally, 259 DNA extracts from blood samples of healthy blood donors were taken as references for local gene distribution.

Table 1 Scoring according to the histological features described by Kleiner et al[15].
DefinitionScore
Steatosis
Grade
< 5%0
5%-33%1
33%-66%2
> 66 %3
Localization
Zone 30
Zone 21
Zone 12
Azonal3
Panacinar4
Type
MacrovesiclesIn %
MicrovesiclesIn %
Mixed
Inflammation
Lobular
No foci0
< 2 foci1
2-4 foci2
> 4 foci3
Portal
No inflammation0
Minimal1
Mild2
Moderate3
Severe4
Fibrosis
None0
Mild/moderate1
Periportal or perisinusoidal2
Bridging fibrosis3
Cirrhosis4
Liver cell damage
Ballooning
None0
Moderate1
Severe2
Mallory bodies
None0
Moderate1
Severe2
Automatic DNA extraction from formalin fixed and paraffin embedded biopsies

Extraction of DNA from 363 formalin fixed and paraffin embedded (FFPE) biopsies was performed from three 7 μm-microtome sections after deparaffinization and proteinase K treatment, as previously described[30]. Then, DNA was purified by means of magnetic bead technology (FormaPure™ Kit of Agentcourt, Beverly MA, USA). All DNA purification steps were carried out by the BioMek FX laboratory automatic workstation (Beckman Coulter, USA) according to the work file and recommendations of Agentcourt.

Furthermore, DNA from 259 blood samples was prepared by the robotic workstation using the Genfind™ Kit of Agentcourt according to the manufacturer’s instructions.

Cloning of reference sequences into pBluescript

For construction of a reference system with sequences of the Pro12Ala locus (rs1805192) carrying either the mutation or the wild type sequence of PPARγ2 gene we used oligonucleotides comprising the proline or alanine encoding sequences (Figure 1). These oligonucleotides were dimerized in 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L Tris pH 7.4, creating EcoRI and SpeI compatible overhangs and inserted into pBluescript SKII (+) (Stratagene, Texas, USA) by the respective restriction sites (Figure 1).

Figure 1
Figure 1 Cloning strategy and validation of a reference system. A: Cloning strategy for the generation of a reference system. The Pro12Ala mutation locus of the peroxisome proliferator-activated receptor-γ gene was synthesized as the proline or the alanine encoding oligonucleotides sequence. The proline or the alanine codon is indicated in yellow. The chemically synthesized oligonucleotide dimers, flanked by the overhangs of the EcoRI and the SpeI restriction sites (red/green), respectively, were used for insertion into pBluescript SKII plasmids; B: Real-time polymerase chain reaction (PCR) of the Pro12Ala locus depending on different copy numbers of reference sequences. Real-time PCR was performed using the LNA probes (Table 2) specific for the proline encoding sequence or the alanine encoding allele, respectively. 106, 105, 104, 103, 102, 10 copies of the plasmid reference sequences encoding either the Ala12 or the Pro12 locus were each diluted in 10 ng salmon sperm DNA and used for LNA probe based real-time PCR assays. Up to 10 copies per ng total DNA of both reference sequences were efficiently detected by the corresponding LNA probe labelled either by Hex or Fam fluorochrome.
Table 2 Real-time polymerase chain reaction primers and probes.
NameOligonucleotide sequencePCR application
Plas A primer5'-CCGCTCTAGAACTAGTGAAGGAA-3'Reference DNA
Plas S primer5'-ACTCACTATAGGGCGAATTGG-3'
PPARγ A primer5'-TTACCTTGTGATATGTTTGCAGACA-3'Target DNA
PPARγ mis primer5'-GTTATGGGTGAAACTCTTGGAGA-3'
TM LNA probe wt5'-6FAM-CTATTGACCCAGAAAGC--BHQ1Target and reference DNA
TM LNA probe mut5'-YAK-CTATTGACGCAGAAAGC--BHQ1
Allelic discrimination of the PPARγ2 by real-time PCR

The Pro12Ala exchange of the PPARγ gene (rs1801282) was examined in DNA from normal blood donors and patients by a Taqman probe associated real-time PCR. Genomic DNA was amplified by real-time PCR in a total volume of 10 μL using the Eppendorf MasterMix (Hamburg, Germany) and 0.3 μL of each primer (10 μmol/L). The Plas A and Plas S primer set was used for amplification of reference plasmids; the PPARγ A and PPARγ mis primer set was used for genomic DNA samples (Table 2).

Allelic discrimination was achieved by adding 0.4 μL of 2.5 μmol/L LNA probes (TIB Molbiol, Berlin, Germany) recognizing the wild type and the mutant variant of the Pro12Ala locus of the PPARγ2 gene (Table 2). In parallel to the allelic Pro12Ala discrimination, plasmid reference sequences diluted from 105 to 10 copies in herring’s sperm DNA (1 ng/mL) were applied to all assays as positive controls. Amplification and analyses were accomplished by the following cycling conditions using a MX3000P qPCR System of Stratagene (Texas, USA): initial denaturation at 95°C for 2 min, following 50 cycles 95°C for denaturation, 55°C for annealing, 65°C for extension, each step lasting 20 s.

Statistical analysis

Pro12Ala distribution was evaluated using the SPSS software 17 of IBM® (Chicago, USA). Significance of cross-classification was calculated by the Fisher’s exact test. Odds ratios were used to describe the risk of disease progression.

RESULTS

Prominent occurence of the PPARγalanine variant in patients with fatty liver disease

In order to detect the Ala12Pro polymorphism in patients with fatty liver disease we established an assay using locked nucleotide acid (LNA) probes for allelic discrimination. For this purpose, both variants of the Ala12Pro locus were cloned (Figure 1A) and the sequences, encoding either the proline or the alanine variant, were used as reference sequences for efficient allele detection and discrimination. The LNA probe hybridization assay linked to real-time PCR efficiently detected both, the proline wild type and the alanine mutated variant of the PPARγ gene (Figure 1B). Up to 10 copies of each reference sequence were successfully proven. In addition to the high sensitivity, application of the reference sequences attested that the LNA probe based PCR assay was highly specific, enabling the differentiation of the alanine and the proline encoding sequence.

In the 259 healthy blood donors the assay accounted for 1.5% homozygous variants carrying the alanine encoding sequence (Figure 2). Analyses of the Pro12Ala distribution in the collective of patients with fatty liver disease (n = 363) revealed an increased incidence of the alanine mutant (3%) compared to the healthy population (1.5%). However, the difference was not statistically significant.

Figure 2
Figure 2 Frequency of the Pro12Ala polymorphism in patients with non-alcoholic fatty liver disease (n = 263), alcoholic fatty liver disease (n = 100) and in healthy blood donors (n = 259). The wild type allele, which is the Pro allele, is indicated in grey, the heterozygous genotype (Pro/Ala) in black and the homozygous Ala/Ala mutant in pale-grey. Genotype analyses revealed a higher prevalence of the homozygous Ala/Ala genotype in non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD) patients.

In order to identify the association of enhanced alanine occurrence, clinical data of patients with fatty liver disease were considered, revealing that 263 patients suffered from NAFLD, whereas fatty liver disease was linked to alcohol consumption in 100 cases (Table 3). In both cohorts, 12%-14% of the subjects were diabetes positive.

Table 3 Occurrence of diabetes mellitus in patients with fatty liver disease n (%).
No C2 (NAFLD)C2 (AFLD)
Diabetes mellitus positive33 (12.5)14 (14)
Diabetes mellitus negative144 (54.8)51 (51)
ND86 (32.7)35 (35)
Total263 (100)100 (100)

Furthermore, we analyzed the transaminase values in the two cohorts, showing a GPT/GOT ratio of 2 in NAFLD patients, but a GPT/GOT ratio of 1 in sera of AFLD patients (Figure 3). The high GPT values in NAFLD patients are in accordance with numerous reports, characterizing the progress of steatohepatitis due to non-alcoholic steatosis in comparison to alcoholic steatosis[31]. With respect to the distribution of the Pro12 and the Ala12 alleles in these two cohorts, we found that in both the AFLD and the NAFLD collective the frequency of the alanine genotype was higher (about 3%) compared to the healthy population (Figure 2).

Figure 3
Figure 3 GOT and GPT levels in sera of non-alcoholic fatty liver disease (A) or alcoholic fatty liver disease (B) patients carrying the Ala mutated allele or the Pro wild type allele of the peroxisome-proliferator-activated receptor-γ2 gene. NAFLD: Non-alcoholic fatty liver disease; AFLD: Alcoholic fatty liver disease. Dots (●) indicate outliers that are not included between the whiskers.
Association of the alanine allele with inflammation and fibrosis in fatty liver disease

We next addressed the question whether the elevated incidence of the alanine allele in the population of fatty liver diseases is associated with the grade of steatosis, ballooning, steatohepatitis, or liver fibrosis (Table 4).

Table 4 Histological features in the fatty liver diseases cohort.
Cases of fatty liver disease (n = 363)n (%)
Steatosis
S09 (2.5)
S1169 (46.6)
S284 (23.1)
S3101 (27.8)
Ballooning
No ballooning164 (45.2)
Stage 1124 (34.2)
Stage 275 (20.7)
Inflammation
G04 (1.1)
G1217 (59.8)
G2113 (31.1)
G329 (8)
Fibrosis
F097 (26.7)
F1116 (32)
F266 (18.2)
F356 (15.4)
F428 (7.7)

The degree of steatosis is traditionally classified into mild (< 30%), moderate (30%-60%), and severe (> 60%). More than 50% of the patients with NAFLD and AFLD had developed severe steatosis, however, in patients carrying the alanine allele severe steatosis occurred slightly more often. Pronounced ballooning was observed likewise often in NAFLD patients with the mutant or the wild type isoform of the PPARγ2 (Table 5).

Table 5 Occurrence of the wt and the mutated form of peroxisome proliferator-activated receptor-γ2 depending on the grade of steatosis and ballooning n (%).
Steatosis
Ballooning
NAFLD
AFLD
NAFLD
AFLD
TotalModerate (0-1)Severe (2-3)TotalModerate (0-1)Severe (2-3)Total-+Total-+
Allelic discrimination
Mutation66 (100)27 (41)39 (59)26 (100)12 (46)14 (54)66 (100)28 (42)38 (58)26 (100)19 (73)7 (27)
Wild type460 (100)229 (50)231 (50)174 (100)88 (51)86 (49)460 (100)206 (45)254 (55)174 (100)121 (70)53 (30)
P value0.1120.4170.4110.454
Odds ratio (CI)1.43 (0.85-2.42)1.19 (0.52-2.73)1.10 (0.65-1.85)0.84 (0.33-2.12)

Histological scoring for inflammatory alterations and fibrosis according to the recommendations described by Kleiner et al[15] (Table 1) revealed that in most of the patients steatosis was accompanied by moderate, mild or severe steatohepatitis. In particular, in patients suffering from AFLD steatohepatitis has passed over to fibrosis in 71% of the cases.

Whereas in NAFLD patients inflammation was not significantly associated with the allelic incidence, in AFLD patients the frequency of the Ala12 variant of the PPARγ2 gene was significantly increased when prominent inflammation had occurred (P = 0.028). The higher risk of AFLD patients developing several inflammatory processes ending in liver fibrosis was also shown by elevated Odds ratios (Odd inflammation = 2.50, CI: 1.05-5.90 and Odd fibrosis = 2.48, CI: 0.81-7.53) (Table 6).

Table 6 Occurrence of the wt and mutated form of the peroxisome proliferator-activated receptor-γ2 depending on the grade of inflammation and fibrosis n (%).
Inflammation
Fibrosis
NAFLD
AFLD
NAFLD
AFLD
TotalModerate (0-1)Severe (2-3)TotalModerate (0-1)Severe (2-3)TotalF0-F1F2-F4TotalF0-F1F2-F4
Allelic discrimination
Mutation66 (100)38 (58)28 (42)26 (100)9 (35)17 (65)66 (100)47 (71)19 (29)26 (100)4 (15)22 (85)
Wild type460 (100)296 (64)164 (36)174 (100)99 (57)75 (43)460 (100)327 (71)133 (29)174 (100)54 (31)120 (69)
P value0.1750.0280.5550.075
Odds ratio (CI)1.33 (0.79-2.25)2.50 (1.05-5.90)0.99 (0.56-1.76)2.48 (0.81-7.53)
DISCUSSION

FLD has a high incidence of approximately 20% worldwide and is regarded as a major cause of liver-related morbidity and mortality due to its risk of progression into cirrhosis or hepatocellular carcinoma. Since the transcription factor PPARγ as been shown to be markedly involved in adipogenesis, hepatic lipid storage and metabolism, we first analyzed the frequency of the Pro12Ala polymorphism of the PPARγ gene in a German cohort of patients with FLD compared to German healthy blood donors. A highly sensitive and robust test was established which was certain to distinguish the Ala and the Pro alleles, even though only low copy numbers from some FFPE biopsies might be available. The genotype distribution (77.5% wt, 21.2% heterozygous, and 1.5% homozygous Ala/Ala mutants) in the collective of the healthy blood donors resembles previous data collected on more than 600 Caucasians by Yen et al[32] and Ghoussaini et al[33]. This genotype distribution in Caucasians, however, differs from the Asian or African frequency, in which less Ala alleles of the PPARγ occur[23,32]. In contrast to the data of healthy blood donors, PCR analyses of DNA from subjects with FLD revealed an increased frequency of the homozygous Ala-subtype up to almost 3.5% in both the AFLD and the NAFLD collectives. Recent meta-analyses summarized data of the Pro12Ala polymorphism in patients with diabetes and identified the mutated Ala variant as a protection factor of diabetes type 2[34-36]. The malfunctioning Ala variant was also shown to be associated with coronary heart disease[37] and with obesity indicated by significant higher BMI in homozygous Ala carriers than in subjects expressing the heterozygous or the wild type PPARγ2 form[38,39]. Although in some reports higher insulin sensitivity and BMI could not be confirmed[40,41], a comprehensive study on 1170 British patients with coronary heart disease[42] and a meta-analysis including 19 136 subjects clearly identified the Ala carriers as individuals with significant higher BMI[43]. Additionally, cholesterol, LDL-cholesterol and apolipoprotein B concentrations are elevated in Ala carriers[42,44]. Therefore, this Ala-associated hyperlipidemia is assumed to be a reason for the 2-fold higher incidence of the Ala genotype in patients with FLD compared to healthy blood donors. Since free fatty acids are shown to be involved in upregulation of Fas/CD-95 death receptor[45], enhanced levels of circulating fatty acids due to impaired PPARγ function in Ala/Ala patients may result in apoptosis and inflammatory processes. In contrast to NAFLD, where no or only a moderate link of inflammatory progression to the Pro12Ala polymorphism was shown, a prominent risk of developing steatohepatitis was observed in AFLD patients carrying the Ala allele. This difference in the associated frequency of the Ala variant encoding the minor active PPARγ2 form[23] argues for a divergent role of the PPARγ2 in mechanisms of AFLD and NAFLD progression. The PPARγ2 isoform is upregulated by phosphatidylinositol 3-kinase activation in response to free fatty acids or by insulin[46]. In post-ischemic liver injury and also in alcohol-induced fibrosis, the PPARγ1 andγ2 variants were shown to be downregulated and to function protectively[47,48]. Therefore, the 2-3-fold higher risk of ALFD patients, but not of NAFLD patients, to develop inflammatory and fibrotic progression if they carry the malfunctioning Ala variant of PPARγ2, emphasizes a more prominent anti-inflammatory impact of the PPARγ2 in AFLD than in NAFLD wt-carriers.

The anti-inflammatory action of PPARγ was also demonstrated by previous studies on hepatic stellate cells[49,50], which take centre stage of sinusoidal liver fibrosis due to their tremendous matrix production and secretion of pro-inflammatory and pro-fibrotic mediators after myofibroblastic transition in chronic liver injury[16]. The authors show that PPARγs repressed in myofibroblastic hepatic stellate cells. Additionally, the inflammatory chemokine expression by hepatic stellate cells is markedly inhibited in response to the activation of PPARγ by the agonistic ligand glitazone[49,50]. Taken into account that the malfunctioning Ala variant is associated with a higher risk of progression into steatohepatitis in AFLD patients these results lead to the suggestion that in particular inflammation and fibrosis of AFLD wt-patients can be attenuated by a treatment with PPARγ thiazolidinedione ligands such as rosiglitazone and pioglitazone. Patients with NASH, however, may benefit from glitazone therapy by other mechanisms like improved insulin sensitivity, decreased hyperlipidemia and impeded steatosis as a result of the therapeutic approach[51,52].

In conclusion, our data of a comprehensive study of the Pro12Ala polymorphism on biopsies with FLD, well classified concerning inflammatory and fibrotic alterations, revealed for the first time an association of the Pro12Ala polymorphism with the risk of developing ASH and suggests a more prominent anti-inflammatory influence of the PPARγ2 on progression of human AFLD than on NAFLD. Therefore, the Pro12Ala polymorphism should be studied on an expanded cohort of AFLD patients, in order to be later integrated in a panel of genetic markers applied for future improved prognosis of disease progression.

COMMENTS
Background

Fatty liver diseases are becoming a common cause of chronic liver diseases in the Western countries encountered in about 20% of the general adult and child population. The spectrum of fatty liver diseases (FLD) independently of causative agents ranges from simple steatosis to steatohepatitis, which can progress to liver fibrosis ending up in cirrhosis or hepatocellular carcinoma. Members of the peroxisome proliferator-activated receptors (PPAR) seem to play a key role in the pathophysiology of FLD by modulating increased glucose uptake and hepatic triglyceride accumulation, but also perform anti-inflammatory signals when steatohepatitis has occurred. Since the transcription factor PPARγ has been shown to be markedly involved in adipogenesis, hepatic lipid storage and metabolism, we analyzed the frequency of the Pro12Ala polymorphism of the PPARγ gene in a German cohort of patients with FLD compared to German healthy blood donors.

Research frontiers

Due to its high impact as an insulin-sensitising transcription factor involved in adipogenesis and lipogenesis, the occurrence of single nucleotide polymorphisms (SNP) in the PPARγ gene was recently addressed by numerous reports studying subjects with insulin resistance, type 2 diabetes, arteriosclerosis, and hypertension. A prevalent SNP association with impaired lipid homeostasis was observed in terms of the N-terminal proline alanine exchange (Pro12Ala) of the extra domain in the PPARγ2 variant. The authors analyzed the frequency of the Pro12Ala polymorphism in the PPARγ gene by a highly sensitive LNA-probe based polymerase chain reaction approach in a total of 622 subjects of a Caucasian population, suffering from fatty liver disease (n = 359) or healthy blood donors (n = 263). In agreement with reports showing a high Ala allele prevalence in patients with impaired lipid metabolism in obese and adipose patient, the Ala allele also occurs more often in FLD patients than in the healthy control group. Interestingly, the interpretation and linkage of the allele frequency to histological evaluation and clinical data demonstrates a prominent risk in alcoholic fatty liver disease (AFLD) patients bearing the Ala allele to develop severe steatohepatitis and fibrosis.

Innovations and breakthroughs

In order to detect the Ala12Pro polymorphism in patients with fatty liver disease the authors established an assay using locked nucleotide acid probes for allelic discrimination. The authors' data of a comprehensive study of the Pro12Ala polymorphism on biopsies with FLD, well classified concerning inflammatory and fibrotic alterations, revealed in patients with AFLD, but not with non-alcoholic fatty liver disease (NAFLD), an significant association of the Pro12Ala polymorphism with the risk to develop steatohepatitits. Therefore, PPARγ is suggested to exert a higher anti-inflammatory impact in progression of human AFLD than NAFLD.

Applications

The association of the Pro12Ala polymorphism with the risk of developing inflammatory progression in patients with AFLD suggests a more prominent influence of PPARγ2 on progression of human AFLD than on NAFLD. Therefore, the Pro12Ala polymorphism should be studied on an expanded cohort of AFLD patients, in order to be later integrated in a panel of genetic markers applied for future improved prognosis of disease progression and therapy planning.

Peer review

This review article provides an overview of SLE-related gastrointestinal system involvements, and there are only few review article in the international literatures in recent years.

Footnotes

Peer reviewer: Teng-Yu Lee, MD, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taichung Veterans General Hospital, 160, Sec. 3, Taichung Harbor Road, Taichung 407, Taiwan, China

S- Editor Wang YR L- Editor O’Neill M E- Editor Zheng XM

References
1.  Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy SM, Hobbs HH. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology. 2004;40:1387-1395.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Vuppalanchi R, Chalasani N. Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: Selected practical issues in their evaluation and management. Hepatology. 2009;49:306-317.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Angulo P. Nonalcoholic fatty liver disease. N Engl J Med. 2002;346:1221-1231.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Clark JM, Diehl AM. Defining nonalcoholic fatty liver disease: implications for epidemiologic studies. Gastroenterology. 2003;124:248-250.  [PubMed]  [DOI]  [Cited in This Article: ]
5.  Torres DM, Harrison SA. Diagnosis and therapy of nonalcoholic steatohepatitis. Gastroenterology. 2008;134:1682-1698.  [PubMed]  [DOI]  [Cited in This Article: ]
6.  Brunt EM. Alcoholic and nonalcoholic steatohepatitis. Clin Liver Dis. 2002;6:399-420, vii.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Burt AD, Mutton A, Day CP. Diagnosis and interpretation of steatosis and steatohepatitis. Semin Diagn Pathol. 1998;15:246-258.  [PubMed]  [DOI]  [Cited in This Article: ]
8.  Monroe PS, Baker AL. Alcoholic hepatitis: update on recognition and management. Postgrad Med. 1981;69:32-43.  [PubMed]  [DOI]  [Cited in This Article: ]
9.  Crabb DW. Alcohol deranges hepatic lipid metabolism via altered transcriptional regulation. Trans Am Clin Climatol Assoc. 2004;115:273-287.  [PubMed]  [DOI]  [Cited in This Article: ]
10.  Browning JD, Horton JD. Molecular mediators of hepatic steatosis and liver injury. J Clin Invest. 2004;114:147-152.  [PubMed]  [DOI]  [Cited in This Article: ]
11.  Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology. 2006;43:S99-S112.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  Day CP. From fat to inflammation. Gastroenterology. 2006;130:207-210.  [PubMed]  [DOI]  [Cited in This Article: ]
13.  Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998;114:842-845.  [PubMed]  [DOI]  [Cited in This Article: ]
14.  Feldstein AE, Gores GJ. Apoptosis in alcoholic and nonalcoholic steatohepatitis. Front Biosci. 2005;10:3093-3099.  [PubMed]  [DOI]  [Cited in This Article: ]
15.  Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313-1321.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Friedman SL. Mechanisms of disease: Mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol. 2004;1:98-105.  [PubMed]  [DOI]  [Cited in This Article: ]
17.  Evans RM, Barish GD, Wang YX. PPARs and the complex journey to obesity. Nat Med. 2004;10:355-361.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature. 1998;391:82-86.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Reynolds K, Goldberg RB. Thiazolidinediones : beyond glycemic control. Treat Endocrinol. 2006;5:25-36.  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Gavrilova O, Haluzik M, Matsusue K, Cutson JJ, Johnson L, Dietz KR, Nicol CJ, Vinson C, Gonzalez FJ, Reitman ML. Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass. J Biol Chem. 2003;278:34268-34276.  [PubMed]  [DOI]  [Cited in This Article: ]
21.  Meirhaeghe A, Amouyel P. Impact of genetic variation of PPARgamma in humans. Mol Genet Metab. 2004;83:93-102.  [PubMed]  [DOI]  [Cited in This Article: ]
22.  Savage DB. PPAR gamma as a metabolic regulator: insights from genomics and pharmacology. Expert Rev Mol Med. 2005;7:1-16.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Knouff C, Auwerx J. Peroxisome proliferator-activated receptor-gamma calls for activation in moderation: lessons from genetics and pharmacology. Endocr Rev. 2004;25:899-918.  [PubMed]  [DOI]  [Cited in This Article: ]
24.  Zhu Y, Qi C, Korenberg JR, Chen XN, Noya D, Rao MS, Reddy JK. Structural organization of mouse peroxisome proliferator-activated receptor gamma (mPPAR gamma) gene: alternative promoter use and different splicing yield two mPPAR gamma isoforms. Proc Natl Acad Sci USA. 1995;92:7921-7925.  [PubMed]  [DOI]  [Cited in This Article: ]
25.  Tankó LB, Siddiq A, Lecoeur C, Larsen PJ, Christiansen C, Walley A, Froguel P. ACDC/adiponectin and PPAR-gamma gene polymorphisms: implications for features of obesity. Obes Res. 2005;13:2113-2121.  [PubMed]  [DOI]  [Cited in This Article: ]
26.  Tönjes A, Stumvoll M. The role of the Pro12Ala polymorphism in peroxisome proliferator-activated receptor gamma in diabetes risk. Curr Opin Clin Nutr Metab Care. 2007;10:410-414.  [PubMed]  [DOI]  [Cited in This Article: ]
27.  He W. PPARgamma2 Polymorphism and Human Health. PPAR Res. 2009;2009:849538.  [PubMed]  [DOI]  [Cited in This Article: ]
28.  Sharma AM, Staels B. Review: Peroxisome proliferator-activated receptor gamma and adipose tissue--understanding obesity-related changes in regulation of lipid and glucose metabolism. J Clin Endocrinol Metab. 2007;92:386-395.  [PubMed]  [DOI]  [Cited in This Article: ]
29.  Neuschwander-Tetri BA. Nonalcoholic steatohepatitis and the metabolic syndrome. Am J Med Sci. 2005;330:326-335.  [PubMed]  [DOI]  [Cited in This Article: ]
30.  Odenthal M, Koenig S, Farbrother P, Drebber U, Bury Y, Dienes HP, Eichinger L. Detection of opportunistic infections by low-density microarrays: a diagnostic approach for granulomatous lymphadenitis. Diagn Mol Pathol. 2007;16:18-26.  [PubMed]  [DOI]  [Cited in This Article: ]
31.  Harrison SA, Oliver D, Arnold HL, Gogia S, Neuschwander-Tetri BA. Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease. Gut. 2008;57:1441-1447.  [PubMed]  [DOI]  [Cited in This Article: ]
32.  Yen CJ, Beamer BA, Negri C, Silver K, Brown KA, Yarnall DP, Burns DK, Roth J, Shuldiner AR. Molecular scanning of the human peroxisome proliferator activated receptor gamma (hPPAR gamma) gene in diabetic Caucasians: identification of a Pro12Ala PPAR gamma 2 missense mutation. Biochem Biophys Res Commun. 1997;241:270-274.  [PubMed]  [DOI]  [Cited in This Article: ]
33.  Ghoussaini M, Meyre D, Lobbens S, Charpentier G, Clément K, Charles MA, Tauber M, Weill J, Froguel P. Implication of the Pro12Ala polymorphism of the PPAR-gamma 2 gene in type 2 diabetes and obesity in the French population. BMC Med Genet. 2005;6:11.  [PubMed]  [DOI]  [Cited in This Article: ]
34.  Kadowaki T, Hara K, Kubota N, Tobe K, Terauchi Y, Yamauchi T, Eto K, Kadowaki H, Noda M, Hagura R. The role of PPARgamma in high-fat diet-induced obesity and insulin resistance. J Diabetes Complications. 2002;16:41-45.  [PubMed]  [DOI]  [Cited in This Article: ]
35.  Hara K, Okada T, Tobe K, Yasuda K, Mori Y, Kadowaki H, Hagura R, Akanuma Y, Kimura S, Ito C. The Pro12Ala polymorphism in PPAR gamma2 may confer resistance to type 2 diabetes. Biochem Biophys Res Commun. 2000;271:212-216.  [PubMed]  [DOI]  [Cited in This Article: ]
36.  Stumvoll M, Häring H. The peroxisome proliferator-activated receptor-gamma2 Pro12Ala polymorphism. Diabetes. 2002;51:2341-2347.  [PubMed]  [DOI]  [Cited in This Article: ]
37.  Dallongeville J, Iribarren C, Ferrières J, Lyon L, Evans A, Go AS, Arveiler D, Fortmann SP, Ducimetière P, Hlatky MA. Peroxisome proliferator-activated receptor gamma polymorphisms and coronary heart disease. PPAR Res. 2009;2009:543746.  [PubMed]  [DOI]  [Cited in This Article: ]
38.  Ek J, Urhammer SA, Sørensen TI, Andersen T, Auwerx J, Pedersen O. Homozygosity of the Pro12Ala variant of the peroxisome proliferation-activated receptor-gamma2 (PPAR-gamma2): divergent modulating effects on body mass index in obese and lean Caucasian men. Diabetologia. 1999;42:892-895.  [PubMed]  [DOI]  [Cited in This Article: ]
39.  Valve R, Sivenius K, Miettinen R, Pihlajamäki J, Rissanen A, Deeb SS, Auwerx J, Uusitupa M, Laakso M. Two polymorphisms in the peroxisome proliferator-activated receptor-gamma gene are associated with severe overweight among obese women. J Clin Endocrinol Metab. 1999;84:3708-3712.  [PubMed]  [DOI]  [Cited in This Article: ]
40.  Deeb SS, Fajas L, Nemoto M, Pihlajamäki J, Mykkänen L, Kuusisto J, Laakso M, Fujimoto W, Auwerx J. A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat Genet. 1998;20:284-287.  [PubMed]  [DOI]  [Cited in This Article: ]
41.  Schäffler A, Barth N, Schmitz G, Zietz B, Palitzsch KD, Schölmerich J. Frequency and significance of Pro12Ala and Pro115Gln polymorphism in gene for peroxisome proliferation-activated receptor-gamma regarding metabolic parameters in a Caucasian cohort. Endocrine. 2001;14:369-373.  [PubMed]  [DOI]  [Cited in This Article: ]
42.  Swarbrick MM, Chapman CM, McQuillan BM, Hung J, Thompson PL, Beilby JP. A Pro12Ala polymorphism in the human peroxisome proliferator-activated receptor-gamma 2 is associated with combined hyperlipidaemia in obesity. Eur J Endocrinol. 2001;144:277-282.  [PubMed]  [DOI]  [Cited in This Article: ]
43.  Masud S, Ye S. Effect of the peroxisome proliferator activated receptor-gamma gene Pro12Ala variant on body mass index: a meta-analysis. J Med Genet. 2003;40:773-780.  [PubMed]  [DOI]  [Cited in This Article: ]
44.  Meirhaeghe A, Fajas L, Helbecque N, Cottel D, Auwerx J, Deeb SS, Amouyel P. Impact of the Peroxisome Proliferator Activated Receptor gamma2 Pro12Ala polymorphism on adiposity, lipids and non-insulin-dependent diabetes mellitus. Int J Obes Relat Metab Disord. 2000;24:195-199.  [PubMed]  [DOI]  [Cited in This Article: ]
45.  Feldstein AE, Canbay A, Guicciardi ME, Higuchi H, Bronk SF, Gores GJ. Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice. J Hepatol. 2003;39:978-983.  [PubMed]  [DOI]  [Cited in This Article: ]
46.  Edvardsson U, Ljungberg A, Oscarsson J. Insulin and oleic acid increase PPARgamma2 expression in cultured mouse hepatocytes. Biochem Biophys Res Commun. 2006;340:111-117.  [PubMed]  [DOI]  [Cited in This Article: ]
47.  Kuboki S, Shin T, Huber N, Eismann T, Galloway E, Schuster R, Blanchard J, Zingarelli B, Lentsch AB. Peroxisome proliferator-activated receptor-gamma protects against hepatic ischemia/reperfusion injury in mice. Hepatology. 2008;47:215-224.  [PubMed]  [DOI]  [Cited in This Article: ]
48.  Tomita K, Azuma T, Kitamura N, Nishida J, Tamiya G, Oka A, Inokuchi S, Nishimura T, Suematsu M, Ishii H. Pioglitazone prevents alcohol-induced fatty liver in rats through up-regulation of c-Met. Gastroenterology. 2004;126:873-885.  [PubMed]  [DOI]  [Cited in This Article: ]
49.  Marra F, Efsen E, Romanelli RG, Caligiuri A, Pastacaldi S, Batignani G, Bonacchi A, Caporale R, Laffi G, Pinzani M. Ligands of peroxisome proliferator-activated receptor gamma modulate profibrogenic and proinflammatory actions in hepatic stellate cells. Gastroenterology. 2000;119:466-478.  [PubMed]  [DOI]  [Cited in This Article: ]
50.  Miyahara T, Schrum L, Rippe R, Xiong S, Yee HF Jr, Motomura K, Anania FA, Willson TM, Tsukamoto H. Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem. 2000;275:35715-35722.  [PubMed]  [DOI]  [Cited in This Article: ]
51.  Ratziu V, Giral P, Jacqueminet S, Charlotte F, Hartemann-Heurtier A, Serfaty L, Podevin P, Lacorte JM, Bernhardt C, Bruckert E. Rosiglitazone for nonalcoholic steatohepatitis: one-year results of the randomized placebo-controlled Fatty Liver Improvement with Rosiglitazone Therapy (FLIRT) Trial. Gastroenterology. 2008;135:100-110.  [PubMed]  [DOI]  [Cited in This Article: ]
52.  Stumvoll M, Häring HU. Glitazones: clinical effects and molecular mechanisms. Ann Med. 2002;34:217-224.  [PubMed]  [DOI]  [Cited in This Article: ]