Association of Genetic Variation in Adaptor Protein APPL1/APPL2 Loci with Non-Alcoholic Fatty Liver Disease
et al. (2013) Association of Genetic Variation in Adaptor Protein APPL1/APPL2 Loci with Non-
Alcoholic Fatty Liver Disease. PLoS ONE 8(8): e71391. doi:10.1371/journal.pone.0071391
Association of Genetic Variation in Adaptor Protein APPL1/APPL2 Loci with Non-Alcoholic Fatty Liver Disease
Michelangela Barbieri 0
Antonietta Esposito 0
Edith Angellotti 0
Maria Rosaria Rizzo 0
Raffaele Marfella 0
Giuseppe Paolisso 0
Massimo Federici, University of Tor Vergata, Italy
0 Department of Medical , Surgical, Neurological , Metabolic and Geriatric Sciences, Second University of Naples , Naples , Italy
The importance of genetics and epigenetic changes in the pathogenesis of non alcoholic fatty liver disease (NAFLD) has been increasingly recognized. Adiponectin has a central role in regulating glucose and lipid metabolism and controlling inflammation in insulin-sensitive tissues and low adiponectin levels have been linked to NAFLD. APPL1 and APPL2 are adaptor proteins that interact with the intracellular region of adiponectin receptors and mediate adiponectin signaling and its effects on metabolism. The aim of our study was the evaluation of a potential association between variants at APPL1 and APPL2 loci and NAFLD occurrence. The impact on liver damage and hepatic steatosis severity has been also evaluated. To this aim allele frequency and genotype distribution of APPL1- rs3806622 and -rs4640525 and APPL2-rs 11112412 variants were evaluated in 223 subjects with clinical diagnosis of NAFLD and compared with 231 healthy subjects. The impact of APPL1 and APPL2 SNPs on liver damage and hepatic steatosis severity has been also evaluated. The minor-allele combination APPL1-C/APPL2-A was associated with an increased risk of NAFLD (OR = 2.50 95% CI 1.45-4.32; p,0.001) even after adjustment for age, sex, body mass index, insulin resistance (HOMA-IR), triglycerides and adiponectin levels. This allele combination carrier had higher plasma alanine aminotransferase levels (Diff = 15.08 [7.60-22.57] p = 0.001) and an increased frequency of severe steatosis compared to the reference allele combination (OR = 3.88; 95% CI 1.582-9.531; p,0.001). In conclusion, C-APPL1/A-APPL2 allele combination is associated with NAFLD occurrence, with a more severe hepatic steatosis grade and with a reduced adiponectin cytoprotective effect on liver.
Funding: This work was supported by the Second University of Naples. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Nonalcoholic fatty liver disease (NAFLD) is the most common
chronic liver disease in the Western population, encompassing a
spectrum of histological injury ranging from pure benign steatosis
to progressive necro inflammation and fibrosis . Altered
adipokine action and increased oxidative stress are candidate
pathogenetic mechanisms in NAFLD . Among the different
adipokines, low adiponectin levels predict severity of liver disease
in NAFLD, even in the absence of diabetes and obesity .
Adiponectin exerts its effects through two membrane receptors,
AdipoR1 and AdipoR2 . Although the post receptor signalling
remains to be elucidated, it seems that adiponectin achieves its
function in the liver via activating 5-AMP-activated protein kinase
(AMPK) and peroxisome proliferator-activated receptor (PPAR)-a
pathways [34,8]. Adaptor protein, PH domain and leucine zipper
containing 1 (APPL1) and Adaptor protein, PH domain and
leucine zipper containing 2 (APPL2) are the first identified adaptor
proteins that interact directly with adiponectin receptors .
They are both highly expressed in insulin target tissues, including
skeletal muscle, liver and adipose tissue and mediate adiponectin
signaling and its effects on metabolism via binding to N terminus
of adiponectin receptors . Adiponectin signaling through
APPL1 is necessary to exert its anti-inflammatory and
cytoprotective effects on endothelial cells . APPL1 also functions in
insulin-signaling pathway  and is an important mediator of
adiponectin dependent insulin sensitization in skeletal muscle and
also acts as a mediator of other signaling pathways by interacting
directly with membrane receptors or signalling proteins, thereby
playing critical roles in cell proliferation, apoptosis, cell survival,
endosomal trafficking, and chromatin remodelling . The
function of APPL2 is not entirely clear even if it was reported to
down regulate the ADIPO R1 signaling . Interestingly, over
expression of APPL1 in mouse hepatocyte cells can activate p38
mitogen-activated protein kinases (MAPK), and adiponectin
treatment could further enhance this effect, suggesting that APPL1
plays a role in adiponectin signaling in liver . Genetic as well as
environmental factors are important in the development of
NAFLD. While it is well known that environmental risk factors
influence the development and progression of NAFLD ,
the contribution of the individual genetic variation to disease
predisposition remains uncertain, despite the fact that several
genes, including PPARa, phosphatidyl ethanolamine
methyltransferase (PEMT), and Patatin like phospholipase-3 (PNPLA3), have
been suggested as potential candidates for either NAFLD
susceptibility or disease progression . Since APPL1 and
APPL2 mediate the effects of adiponectin on target tissues, they
have been considered to be strong candidates in the pathogenesis
of NAFLD. Thus, we hypothesized that genetic variants in APPL1
and APPL2 genes might affect the NAFLD risk through the effects
on adiponectin signaling.
Common single-nucleotide polymorphisms (SNPs) have been
identified in both APPL1 and APPL2 gene. Previous studies found
a significant association between an APPL1 gene variant with
body fat distribution in Type 2 Diabetes Mellitus (T2DM) 
although no association with development of prediabetes
phenotypes  in a healthy metabolically well-characterized population
has been demonstrated. A significant association of APPL2 genetic
variation with overweight and obesity has been also found .
To our knowledge, the contribution of these SNPs to liver lipid
accumulation and liver function has not yet been investigated.
The aim of our study will be the evaluation of a potential
association between the APPL1 and APPL2 loci and the
occurrence and progression of non alcoholic fatty liver disease
(NAFLD). To this aim, allele frequency and genotype distribution
of rs3806622 and rs4640525 of APPL1 and rs 11112412 of APPL2
gene variants were evaluated in subjects with clinical diagnosis of
NAFLD and compared with control healthy subjects. The impact
of APPL1 and APPL2 SNPs on liver damage and hepatic steatosis
severity has been also evaluated.
The APPL1- rs3806622 and -rs4640525 and APPL2-rs
11112412 variants were chosen on the basis of their high MAF
(0. 435, 0.489, 0.193) and previous evidences showing their
significant association with body fat and its distribution and with
cardiovascular risk in type 2 diabetes [22,2425]. Other variants
have been previously studied and found in linkage disequilibrium
Materials and Methods
223 Caucasians subjects (129 males and 94 females; mean age:
55611 years) with clinical diagnosis of NAFLD, living in southern
Italy and referred to our Department, were enrolled. The
diagnosis of NAFLD was established based on the following
inclusion criteria: (1) persistently abnormal levels of aspartate
aminotransferase and alanine aminotransferase (AST/ALT), (2)
evidence of fatty liver using ultrasound imaging techniques, and (3)
screened negative for viral markers (such as HBsAg, anti-HBc,
anti-HBs, anti-HCV, HCV RNA and anti-HIV) .
Additionally, 231 healthy individuals (114 males and 117
females; mean age: 54614 years), matched for sex, age, body mass
index (BMI), ethnicity and life style, without steatosis by
ultrasonography, free of any known major diseases were also
Patients with the following diseases were excluded from the
study: infectious hepatitis (hepatitis B and C, Epstein-Barr virus
infection), autoimmune hepatitis, primary biliary cirrhosis,
sclerosing cholangitis, hemochromatosis, a1-antitrypsin deficiency,
Wilsons disease, drug-induced hepatitis, alcoholic hepatitis, and
excessive alcohol consumption (present or past daily consumption
of more than 20 g alcohol per day). Patients affected by diabetes
were also excluded. Clinical information was obtained by routine
laboratory analysis, history and physical examination. The
maximal alcohol consumption of the study participants was 30 g
per week in men and 20g in woman.
The use of human blood sample and the protocol in this study
were strictly conformed to the principles expressed in the
Declaration of Helsinki and were approved by the Institutional
Ethical Committees of the Second University of Naples. Written
informed consent was obtained from all participants before their
participation in the study.
Non-invasive evaluation of the distribution pattern of liver fatty
infiltration was performed by ecography . The echography
patterns of fatty liver disease appears as bright liver (brightness
and posterior attenuation) with stronger echoes in the hepatic
parenchyma than in the renal parenchyma, vessel blurring and
narrowing of the lumens of the epatic veins in the absence of
findings, which are suggestive of other cronic liver desease. Mild,
moderate or severe steatosis degree was defined according to the
following items : 1. Diffuse enhancement of near field echo in
the hepatic region (stronger than in the kidney and spleen region)
and gradual attenuation of the far field echo. 2. Unclear display of
intra-hepatic lacuna structure. 3. Mild to moderate hepatomegaly
with a round and blunt border. 4. Color Doppler ultrasonography
shows a reduction of the blood flow signal in the liver or it is even
hard to display, but the distribution of blood flow is normal. 5.
Unclear or non-intact display of envelop of right liver lobe and
Mild degree of fatty liver displays item 1 and any one of items
24; moderate fatty liver displays item 1 and any two items of
items 24; severe fatty liver displays items 1 and 5 and any two of
items 24 .
Anthropometric determinations (weight, height, BMI, and
waist/hip ratio) were measured by standard techniques. Plasma
fasting AST, ALT, gamma glutamil transferasi (G-GT), total
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides,
ferritin, glucose, adiponectin, insulin, were determinated by
routine laboratory methods (Roche Diagnostics, Monza (MI),
All individuals were genotyped for rs3806622 and rs4640525
polymorphisms of the APPL1 gene on chromosome 3p21.1-p14.3
and for rs11112412 polymorphism of the APPL2 gene on
chromosome 12q 24.1. Genomic DNA was obtained from blood
lymphocytes collected into EDTA-containing tubes using a
commercial DNA extraction kit (Illustra, GE Healthcare UK
Limited, Buckinghamshire, HP7 9 NA,UK). Reference APPL1
and APPL2 genome sequence were obtained from the NCBI
database (Homo Sapiens Chromosome, GeneBank AB037849 and
AY113704). Primers chosen for amplification were designed using
Primer 3 software. Amplification was performed using the
following primers: APPL1-rs4640525-F:
59TTTGTGGAATTGGTCAGGTG-39; APPL1- rs4640525-R:
APPL1-rs3806622R: 59-TGAGGGCT ACAAG CCTATCCT-39) and
APPL2rs11112412-F (59-ATTCAACAAGGGCACAGTCC -39)
APPL2rs11112412-R (59- CATTGCCAGCGAGTGTTCTA-39).
The Polymerase chain reaction (PCR) was carried out under the
following conditions: 95uC for 5 min, followed by 35 cycles of
95uC for 30 s, 60uC for 30 s, 72uC for 1 min, with final extension
of 72uC for 4 min. Genotyping was performed using restriction
analysis (RFLP) using the Hpy188I enzyme for APPL1 rs4640525,
the BfaI enzyme for APPL1 rs3806622 and Hind III for APPL2
rs11112412 and incubating at 37uC over night. The resulting
products were identified on 4.5% agarose gel.
Laboratory personnel who assessed all genotyping results were
blinded to the samples casecontrol status. Genotyping quality was
examined by a detailed quality control procedure consisting of
95% successful call rate, duplicate calling of genotypes, internal
positive control samples and HardyWeinberg Equilibrium
(HWE) testing. The concordance rate was greater than 99%
based on 10% of duplicate samples for each SNP.
Directly DNA sequencing was use in a subset of 50 individuals
to further confirm the genotypes for each SNP.
Insulin resistance (HOMA-IR) was calculated according to the
homeostasis model assessment (HOMA) (3031): insulin resistance
(IR) = FI x G/22.5 where FI = fasting insulin (mU/ml) and
G = fasting glucose (mmol/l) .
For each SNP, the genotypic and allelic frequencies were
calculated. The Chi square test was used to compare the expected
genotypic frequencies with the actual frequencies observed based
on the HardyWeinberg equilibrium. The difference in genotype
frequency between NAFLD and controls was analyzed by
standard contingency Chi square test. Univariate and multivariate
logistic regression analyses were performed to obtain the crude
and adjusted odds ratios (ORs) for risk of NAFLD and their 95%
confidence intervals (CIs). Age, sex, HOMA (IR), plasma
adiponectin and triglycerides levels and BMI were considered as
potential confounders and were also included in the multivariate
logistic regression models.
Differences between groups were analyzed by ANOVA when
variables were normally distributed; otherwise, the Mann
Whitney U-test was used. The Bonferroni correction was applied
to the analysis of study groups. Associations between genotypes
and metabolic profile parameters were analyzed using the General
Statistical analyses were performed using SPSS software
package. All metabolic parameters are presented as means 6
standard deviation (SD). In order to investigate differences
between the two study groups, sample size was estimated by
GPOWER software. The resulting sample size, estimated
according to a global effect size of 0.25 with type I error of 0.05 and a
power of 95% was 210 patients.
Linkage disequilibrium and allele combination analyses were
performed by use of the Thesias program based on the
stochasticEM algorithm . The Thesias program allows estimation of
both haplotype frequencies and covariable-adjusted haplotype
effects by comparison with a reference haplotype taken as the most
frequent haplotype in the current analyses. A global test of
association between haplotypes and any studied phenotype was
performed by means of a chi-square test with m-1 degree of
freedom in the case of m haplotypes.
Clinical and biochemical characteristics of the study subjects are
reported in Table 1. All subjects (n = 454) were adult (mean
age = 54.9613), overweight, with no difference in gender ratio
(211 F/243 M, x2 = 2.25 p = 0.130). Compared to control
subjects, NAFLD patients had higher occurrence of most of the
risk factors of the metabolic syndrome, including elevated blood
pressure, IR (HOMA) index, fasting plasma glucose, total
cholesterol, triglycerides and decreased HDL-C. In addition,
levels of alanine transaminase (ALT) and aspartate transaminase
(AST) were significantly higher in patients with NAFLD,
compared to controls. No difference in gender distribution was
found in both groups (Table1).
Total Cholesterol (mgl/dl)
HDL Cholesterol (mg/dl)
Systolic Blood Pressure (mmHg)
Diastolic Blood Pressure (mmHg)
In all study groups, the genotype frequencies of all gene variants
studied respected the Hardy-Weinberg equilibrium (p.0.05 for all
the SNP investigated). The genotypic frequencies observed for all
gene variants studied were almost similar to those reported in
previous studies among Caucasians.
A strong linkage disequilibrium between the rs4640525 and
rs3806622 APPL1 gene polymorphisms studied was observed
(D9 = 0.97, r2 = 0.81). Since these two variants would be predicted
to provide identical/nearly identical genotypic information the
rs3806622 variant was excluded from further analyses.
The minor allele frequencies was 0.456 for rs4640525 APPL1
gene polymorphisms and 0.184 for rs11112412 APPL2. The
frequency of APPL1 rs4640525 GG, GC and CC genotypes was
30.4%, 48% and 21.6% respectively. As far as rs11112412 APPL2
gene variant is concerned the frequency of GG, GA and AA
genotypes was 66.3%, 30.6% and 3.1.% respectively.
Association of APPL1 and APPL2 Genes with NAFLD
Genotype distributions of APPL1 and APPL2 polymorphisms in
patients with NAFLD and control subjects and their associations
with NAFLD are summarized in Table 2. A different rs 4640525
APPL1 genotype distribution was found between NAFLD and
healthy control subjects while no differences were found when
genotype distribution of APPL2 gene polymorphism was analyzed.
Indeed, both the C allele at the APPL1 locus and the A allele at
the APPL2 locus were found to be more represented among
NAFLD subjects in comparison to controls.
A logistic regression analysis was used to estimate associations
between the APPL1 and APPL2 variant and the risk of NAFLD
(Table 2). It was found that individuals with at least one copy of
the rs4640525 C allele and rs11112412 APPL2 A allele had
respectively a 1.73 (95% CI: 1.282.32; P = 0.002) and 1.63 (95%
CI: 1.112.41; P = 0.01) fold increased risk compared with those
without this allele. Adjustment for age, BMI, HOMA IR,
x2 = 12.5; p = 0.002
Fishers test: P = 0.001
x 2 = 4.64; p = 0.098
Fishers test: P _ 0.04
Odds ratio (95%CI)
APPL2 polymorphisms on ALT levels was assessed by means of a
General Linear Model ANOVA, including age, IR (HOMA) and
BMI, plasma triglycerides and adiponectin levels as covariates.The
analysis revealed higher plasma ALT levels in C+subjects (GC and
CC genotypes) in comparison to C- subjects (GG genotype):
59.4861.89 vs 46.0262.87 U/L, F = 15.2, p = 0.003, as well as in
APPL2 A+ individuals (GA and AA genotypes) in comparison to
A- individuals (GG genotypes): 61.3962.74 vs 52.3661.95,
F = 7.12, p = 0.008). Comparing the different grading of hepatic
steatosis among the three genotype groups there was an increased
frequency of severe steatosis in NAFLD individuals with
APPL1CC genotype; a trend for individuals with APPL2 AA genotype to
have an higher frequency of severe steatosis was also found; the
differences however did not reach statistical significance (Figure 1).
A logistic regression analysis revealed that individuals with at
least one copy of the rs4640525 C allele and rs11112412 APPL2 A
allele had a 2.17 (95% CI: 1.273.711; P = 0.004) and 1.96 (95%
Data are presented as % (n);
*adjusted for age, BMI, IR HOMA index, plasma adiponectin and triglycerides levels.
triglycerides and adiponectin levels did not appreciably change the
Considering the possibility of interchromosomal interaction,
allele combination analysis of APPL1 and APPL2 variants have
been performed. Allele combination frequencies in NAFLD and
control subjects are shown in Table 3. When the most common
allele combination consisting of the 2 major alleles G-G considered
as reference, as expected the all-minor-allele combination CA was
associated with an increased risk of NAFLD (OD = 2.50 95% CI
1.454.32; p,0.0009) even after adjustment for age, sex, BMI,
HOMA IR, triglycerides and adiponectin levels.
Impact on Quantitative Traits
The impact of genetic variability at APPL1 and APPL2 on AST
and ALT plasma levels was tested in univariate analysis (Table 4).
APPL1 and APPL2 genotypes affected ALT plasma levels,
whereas no difference in AST plasma levels according to
genotypes was observed. Accordingly, the effect of APPL1 and
Odds ratio (95%CI)
Adj Odds ratio (95%CI)*
*adjusted for age, BMI, IR HOMA index, plasma adiponectin and triglycerides levels.
CI: 1.00083.83; P = 0.01) fold increased risk for severe steatosis
Further allele combination-phenotype analyses revealed a
significant association of CA allele combination with ALT plasma
levels and grading of hepatic steatosis after adjustment for age,
BMI, HOMA IR, adiponectin and triglycerides plasma levels.
This allele combination carriers had higher plasma ALT levels
(Diff = 15.08679 [7.6024722.57111] p = 0.000078) and an
increased frequency of severe steatosis compared to the reference
allele combination (Table 5).
In the present study, we investigated the effect of two SNPs at
APPL1 and APPL2 loci in relation to NAFLD occurrence. The
major finding of this study is that the rs4640525-APPL1/
rs11112412-APPL2 CA allele combination is associated with an
increased occurrence of NAFLD after controlling for potential
confounders, such as age, BMI, HOMA index and adiponectin
plasma levels. The analysis also revealed higher plasma ALT levels
and a more severe hepatic steatosis grade for CA allele
NAFLD is recognized as the most common type of chronic liver
disease in Western countries and the leading cause of cryptogenic
cirrhosis . The importance of genetic and epigenetic changes
in the aetiology and pathogenesis of NAFLD has been increasingly
recognized and a large number of SNPs related to NAFLD has
been documented by candidate gene studies [1821,33] To our
APPL2 - rs11112412
*p,0.05 vs GG genotype; 1 p,0.05 vs No carrier.
knowledge, this is the first study providing evidence of an
association between APPL1 and APPL2 genetic variants and
NAFLD in humans.
Although the pathogenesis of NAFLD is not fully elucidated, a
complex interaction between adipokines and cytokines produced
by adipocytes and/or inflammatory cells infiltrating adipose tissue
appears to play a crucial role in Metabolic Syndrome and NAFLD
Adiponectin is the most abundant and adipose-specific
adipokine. There is an evidence that adiponectin decreases
hepatic and systematic IR and attenuates liver inflammation and
fibrosis. Insulin resistance (IR) is a key factor in the pathogenesis of
NAFLD, the latter being considered as the hepatic component of
IR or metabolic syndrome [4,34]. The insulin-sensitizing effect of
adiponectin appears to be mainly through enhancement of fatty
acid oxidation and glucose uptake in muscle and inhibition of
gluconeogenesis in liver , which are mediated by two cell
surface receptors, AdipoR1 and AdipoR2 , by a direct
interaction with the extracellular COOH terminus of these
APPL1 and APPL2 have been suggested as the missing link in
the adiponectin-signaling cascade, transmitting signals from
adiponectin receptors to downstream targets by directly
interacting with the NH2-terminal intracellular region of AdipoR1 and
Adiponectin signaling through APPL1 is necessary to exert its
anti-inflammatory and cytoprotective effects on endothelial cells
. APPL1 also functions in insulin-signaling pathway and is
an important mediator of adiponectin dependent insulin
sensitization in skeletal muscle . Furthermore, over expression of
APPL1 in mouse hepatocyte cells activate p38 MAPK, and
adiponectin treatment further enhance this effect, suggesting that
APPL1 plays a role in adiponectin signaling in liver . The
function of APPL2 is not entirely clear even if recently, it has also
been suggested to play an important role in adiponectin and
insulin signaling as well as the cross-talk between these two
Previous studies have demonstrated a significant association
between APPL1 gene variants and body fat distribution in T2DM
 and a significant association of APPL2 genetic variation with
Diff = 15.08[7.6022.57]
Odds ratio 95%CI)for
*Phenotypic Mean = 55.39; Standard Error = 36.46; Residual Standard Error = 35.74.
overweight and obesity . The association of APPL1 and
APPL2 variant with NAFLD, hepatic function and distribution
pattern of liver fatty infiltration has not been previously explored.
Our results, firstly demonstrate an association of rs4640525APPL1
G/C and rs11112412 -APPL2 GA variants with NAFLD being
the A allele at the APPL2 locus and the C allele at the APPL1
locus more represented among NAFLD subjects in comparison to
controls. Moreover, the CA allele combination was associated with
a 2.5 fold increased risk of NAFLD even after adjustment for age,
sex, BMI, HOMA IR and adiponectin levels.
The mechanism through which genetic variants at APPL1 or
APPL2 locus could influence NAFLD occurrence can not been
confirmed. It is likely that the SNPs investigated, which are placed
in intronic regions, are only genetic markers in strong LD with
other genetic variants having biological effects. Indeed, we can
hypothesize that the SNPs studied may differently affect the
expression levels of APPL1 or APPL2, which leads to an altered
adiponectin activity, a decrease in AMPK activation, hepatic
glucose uptake and free fatty acid (FFAs) oxidation and an increase
in de novo lipogenesis and gluconeogenesis, resulting in
intrahepatic lipid accumulation and fatty liver. The final effect is an
impaired adiponectin cytoprotective effects on liver. In agreement,
in our study both higher rs4640525-C and rs11112412-A NAFLD
risk alleles have been found associated with higher plasma ALT
levels and a more severe hepatic steatosis grade.The effect found
was independent of plasma adiponectin level underlining that,
despite normal or high adiponectin levels, an impaired post
receptor signalling due to APPL1/APPL2 SNPS may alter
adiponectin efficiency and activity. Accordingly, in cultured
myotubes, APPL1 knockdown lowers adiponectin- stimulated
fatty acid oxidation, glucose uptake, and phosphorylation of
AMPK, acetyl-CoA carboxylase (ACC) and p38 . Likewise,
overexpression of wild-type APPL1 enhances basal glucose
transporter type 4 (GLUT4) glucose transporter type
4translocation, while overexpression of dominant negative APPL1 inhibits
basal, insulin- and adiponectin stimulated glucose trasporter type 4
(GLUT4) translocation .
Interestingly, the significant interaction found between APPL1
and APPL2 strongly support the hypothesis that interaction with a
different genetic and/or environmental background may
differently modulate the effect of a given gene in different populations.
APPL2 is an isoform of APPL1 and can form a dimer with APPL1
. APPL2 can negatively regulate adiponectin signaling by
competing with APPL1 in binding to AdipoR1 . Indeed,
suppression of APPL2 can promote adiponectin-stimulated
glucose uptake and fatty acid oxidation .Although, unlike
APPL1, APPL2 does not directly interact with the catalytic subunit
of PI3-kinase and Akt2 which are key kinases in the PI3-kinase
pathway downstream of the insulin receptor , APPL2 can
suppress insulin signalling by inhibiting the interaction between
APPL1 and the components of insulin signaling. Thus, suppression
of APPL2 greatly enhanced the sensitizer effect of adiponectin on
Other SNPs of genes regulating insulin signalling, lipid
metabolism, oxidative stress, inflammation have previously
associated with NAFDL, including APOC3, PNPLA3, FABP1, TNFa,
HF3, PEMT, but the contribution of the individual genetic
variation to diseases predisposition remains uncertain .
Although there is evidence that these genetic factors account for
considerable variability in susceptibility to NAFLD, most studies
have not been well validated by larger replication cohorts. Only
PNPLA3 gene variant has been more extensively examined and
validated by a genome wide association study .
In conclusion, our study demonstrates that C-APPL1/A-APPL2
allele combination is associated with an increase NAFLD
occurrence, with a more severe hepatic steatosis grade and with
a reduced adiponectin cytoprotective effects on liver. Such an
effect is probably due to the combined effect of APPL1 and APPL2
genes on both adiponectin and insulin signaling.
Potential limitations in our study need to be addressed. Firstly,
the diagnosis of NAFLD was primarily based on ultrasonographic
findings. Indeed for ethical reasons, it is very difficult to perform
liver biopsies in an epidemiological survey. Secondly, our study
firstly reported the relationship between SNPs of APPL1 and
APPL2 genes and NAFLD in an Italian population subjects. Thus,
further studies will be necessary for replicating our finding in an
independent larger population group and other races.
Conceived and designed the experiments: GP MB RM EA AE MRR.
Performed the experiments: AE EA MB MRR. Analyzed the data: GP MB
RM EA AE MRR. Wrote the paper: GP MB MRR.
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