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Non-alcoholic fatty liver disease
Genetic variants regulating insulin receptor signalling are associated with the severity of liver damage in patients with non-alcoholic fatty liver disease
  1. P Dongiovanni1,
  2. L Valenti1,
  3. R Rametta1,
  4. A K Daly2,
  5. V Nobili3,
  6. E Mozzi1,
  7. J B S Leathart2,
  8. A Pietrobattista3,
  9. A D Burt2,
  10. M Maggioni4,
  11. A L Fracanzani1,
  12. E Lattuada1,
  13. M A Zappa1,
  14. G Roviaro1,
  15. G Marchesini5,
  16. C P Day2,
  17. S Fargion1
  1. 1Università degli Studi Milano, Policlinico MaRE IRCCS Hospital, Milan, Italy
  2. 2Institute of Cellular Medicine, Newcastle University, UK
  3. 3Liver Unit, “Bambino Gesù” Children's Hospital and Research Center, Rome, Italy
  4. 4Unitá Operative Anatomia patologica, Ospedale Sam Paulo and Policlinico IRCCS Milan, Italy
  5. 5Universitá Alma Mater, Bologna, Italy
  1. Correspondence to Dr L Valenti, Centro per lo Studio delle Malattie del Fegato e del Metabolismo, Department of Internal Medicine, UO Medicina Interna 1B, University of Milano, Ospedale Policlinico Mangiagalli e Regina Elena Fondazione IRCCS, Via F Sforza 35, 20122 Milano, Italy; luca.valenti{at}unimi.it

Abstract

Background/aims The aim of this study was to assess the effect of functional ENPP1(ectoenzyme nucleotide pyrophosphate phosphodiesterase 1)/PC-1 (plasma cell antigen-1) and IRS-1 (insulin receptor substrate-1) polymorphisms influencing insulin receptor activity on liver damage in non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, whose progression is associated with the severity of insulin resistance.

Patients and methods 702 patients with biopsy-proven NAFLD from Italy and the UK, and 310 healthy controls. The Lys121Gln ENPP1/PC-1 and the Gly972Arg IRS-1 polymorphisms were evaluated by restriction analysis. Fibrosis was evaluated according to Kleiner. Insulin signalling activity was evaluated by measuring phosphoAKT levels by western blotting in a subset of obese non-diabetic patients.

Results The ENPP1 121Gln and IRS-1 972Arg polymorphisms were detected in 28.7% and 18.1% of patients and associated with increased body weight/dyslipidaemia and diabetes risk, respectively. The ENPP1 121Gln allele was significantly associated with increased prevalence of fibrosis stage >1 and >2, which was higher in subjects also positive for the 972Arg IRS-1 polymorphism. At multivariate analysis, the presence of the ENPP1 121Gln and IRS-1 972Arg polymorphisms was independently associated with fibrosis >1 (OR 1.55, 95% CI 1.24 to 1.97; and OR 1.57, 95% CI 1.12 to 2.23, respectively). Both polymorphisms were associated with a marked reduction of ∼70% of AKT activation status, reflecting insulin resistance and disease severity, in obese patients with NAFLD.

Conclusions The ENPP1 121Gln and IRS-1 972Arg polymorphisms affecting insulin receptor activity predispose to liver damage and decrease hepatic insulin signalling in patients with NAFLD. Defective insulin signalling may play a causal role in the progression of liver damage in NAFLD.

https://doi.org/10.1136/gut.2009.190801

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    Non-alcoholic fatty liver disease (NAFLD), affecting 20–34% of the US and European populations,1 ,2 is considered to be the hepatic manifestation of the metabolic syndrome. NAFLD is characterised by hepatic insulin resistance,3 and is associated with increased mortality due to cardiovascular and liver diseases, including hepatocellular carcinoma.4 ,5

    Insulin resistance is the key factor in NAFLD pathophysiology and deeply entangled with the progression of liver disease. The progressive form of NAFLD is complicated by inflammation—that is, steatohepatitis (non-alcoholic steatohepatitis; NASH)—which is provoked by peripheral insulin resistance leading to an increased supply of free fatty acids to the liver. This results in oxidative, endoplasmic reticulum and cytokine-mediated stress.6

    The presence of the metabolic syndrome predicts the severity of liver disease.4 ,7 Furthermore, insulin resistance is also associated with liver damage in patients with normal aminotransferases,8 but the causal relationship between insulin resistance and the progression of liver damage remains conflicting.

    Inherited factors play a role in the susceptibility to the metabolic syndrome and NASH. Predisposition to NASH appears to cluster with that for metabolic risk factors within families, and ethnic differences have been reported.9 Single nucleotide polymorphisms (SNPs) in genes involved in inflammation, oxidative stress and fibrogenesis have been associated with the severity of liver damage in NAFLD,10–12 and PNPLA3 SNPs have recently been demonstrated to account for interethnic differences in NAFLD susceptibility,13 but the role of genes which directly regulate insulin receptor activity and signalling has not yet been investigated.

    Binding of insulin to the high affinity insulin receptor (INSR) in hepatocytes leads to insulin receptor autophosphorylation, activation of INSR substrate-1 (IRS-1) and IRS-2 and the downstream kinase AKT, which in turn phosphorylates the transcription factor FOXO1 leading to nuclear exclusion and consequent decreased glucose production and apoptosis.14–16 INSR activity is known to be downregulated in the metabolic syndrome, and, in NASH, FOXO1 expression is increased and resistant to inhibition by hyperinsulinaemia.16 Liver-specific deletion of INSR in mice causes hyperglycaemia and liver failure,17 suggesting that hepatic insulin resistance promotes liver damage both by inducing metabolic alterations and by determining hepatocellular apoptosis, which is pivotal in NASH progression.18

    Functional common SNPs of genes included in the depicted pathway have consistently been reported to influence insulin resistance and the susceptibility to type 2 diabetes. Among them, the PC-1 (plasma cell antigen-1)/ENPP1 (ectoenzyme nucleotide pyrophosphate phosphodiesterase 1) Lys121Gln SNP enhances the interaction between the ENPP1 glycoprotein and INSR, inhibiting INSR activity, and is associated with an increased risk of type 2 diabetes.19 Downstream, the IRS-1 Gly972Arg SNP decreases IRS-1 activity and inhibits INSR autophosphorylation and activity,20 and has repeatedly been associated with increased risk of insulin resistance and diabetes.21

    With the hypothesis that insulin resistance plays a causal role in the progression of liver damage in NAFLD, the aim of this study was to assess the effect of these well-characterised common functional SNPs on the severity of liver disease in a large series of patients with biopsy-proven NAFLD, and to determine their influence on hepatic insulin signalling in a subset of cases.

    Methods

    Subjects

    We considered 702 unrelated patients from Italy and the UK with biopsy-proven NAFLD diagnosed between January 1999 and January 2008, whose DNA samples and complete clinical data were available (out of a larger group of 940 subjects with incomplete information or unavailable DNA samples). These included 240 adult patients from Milan, 328 from Newcastle and 71 children from the Rome centre (tertiary referral centres), submitted to liver biopsy because of persistently abnormal liver enzymes/serum ferritin or a long-lasting history of steatosis associated with severe metabolic abnormalities, and 63 severely obese patients who were found to be affected by NAFLD at routine liver biopsy performed during bariatric surgery. Other causes of liver disease were excluded, including increased alcohol intake (>30/20 g/day for males/females), as confirmed by at least one family member or friend and carboxydesialylated transferrin determination, autoimmune liver diseases (all patients were tested for antinuclear antibodies (ANAs), antismooth muscle antibodies (ASMAs), LKM (liver–kidney microsomal antibodies), AMAs (antimitochondrial antiboies) and immunoglobulins, and in all patients the ANA titre was <1/160), hereditary haemochromatosis, α1-antitrypsin (AAT) deficiency, Wilson disease and viral hepatitis. Part of this group had previously been described.10 Body mass index (BMI) and metabolic parameters, including glucose and lipid levels, ferritin, and liver enzymes (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT)) were available in all patients, and evaluated as previously reported.8 Demographic and clinical features are shown in table 1.

    View this table:
    Table 1

    Demographic and clinical features of patients with non-alcohoic fatty liver disease (NAFLD) and controls

    Controls

    The control group included 310 Italian subjects out of a larger series of 482 blood donors from Northern Italy (64%) without clinical and biochemical evidence of liver and metabolic disease, inflammatory indices within normal limits, and no alcohol abuse. We excluded subjects with ALT >35/30 IU/ml in males/females, GGT >35 IU/ml, BMI >28, abdominal circumference >100 cm, glucose levels ≥100 mg/dl, triglycerides ≥150 mg/dl or a fatty liver index (FLI) >50, a value with high specificity to rule out NAFLD in the general population.22 In contrast, all patients included had an FLI score >50.

    Informed written consent was obtained from each patient and control subject, and the study conforms to the ethical guidelines of the 1975 Declaration of Helsinki.

    Histological assessment

    Tissue sections were stained with H&E, impregnated with silver for the reticulin framework, and stained with trichrome for collagen. Two expert pathologists unaware of clinical and genetic data reviewed all biopsies for fibrosis stage. The presence of NASH was assessed according to Kleiner et al.23 Briefly, histological activity (NAS: NASH activity score) is graded on steatosis (0–3), lobular inflammation (0–3) and hepatocyte ballooning (0–2), whereas liver fibrosis is staged from 0 (absent) to 4 (cirrhosis). The minimum biopsy size was 1.7 cm and the number of portal areas was 10.

    We chose as main outcome a fibrosis stage >1 according to Kleiner et al,23 in an attempt to identify patients with potentially progressive disease at an early stage. However, we also provided a separate analysis for fibrosis >2.

    Genetic analysis

    DNA was extracted from peripheral blood by the phenol–chloroform method. The success rate in extracting DNA was 100% for each study group. The ENPP1 Lys121Gln (rs1044498) and IRS-1 Gly972Arg (rs1801278) SNPs were determined by restriction analysis, as previously described24 ,25 by personnel unaware of the subjects' clinical status. Samples from both patients with NAFLD and controls were included in all batches analysed, and quality controls were performed to verify the reproducibility of the results. Valid genotypic data were obtained for >99% of subjects analysed. Subjects for whom incomplete genetic data were available were excluded from the analyses.

    Evaluation of AKT phosphorylation status

    The activation status of the insulin signalling pathway was estimated by measuring the AKT activation status in a subgroup of 24 patients with severe obesity without diabetes, who were not taking drugs influencing glucose levels and insulin sensitivity, and for whom sufficient frozen biological samples were available. These subjects were younger and had a higher BMI than the rest of the cohort (p<0.05), but their clinical features were not significantly different from those of the other subjects submitted to bariatric surgery included in the study.

    Tissues (10 mg) were lysed in RIPA buffer. Equal amounts of proteins (25 μg) were separated by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred electrophoretically to nitrocellulose membrane (Biorad, Hercules, California, USA). The blot was incubated with anti-phosphoAKT (pAKT; Thr308 and Ser473) (Cell Signaling Technology, Danvers, Massachusetts, USA) and β-actin antibodies (from Santa Cruz Biotechnology, Santa Cruz, California, USA). The gels were densitometrically scanned and images were analysed with the ImageJ analysis software provided by the NIH.16

    Statistical analysis

    The manuscript was prepared in accordance with the STROBE guidelines.26 The sample size to assess the effect of genetic factors on liver fibrosis was calculated on the basis of the expected relative risk of the mutant allele versus the wild-type allele, the desired power and significance. Our sample had a 70–99% power of detecting an odds ratio (OR) of 2.5 for fibrosis >1 in patients with NAFLD, according to genotype frequencies, with a significance of 5%. Results are expressed as means (SD) and considered significant when p<0.05 (two-tailed). Mean values were compared by t test and frequencies by Fisher exact test.

    The association between the ENPP1 and IRS-1 SNPs and the presence of moderate/severe fibrosis (Kleiner stage >1) was evaluated by logistic regression analysis adjusted for confounders (age, BMI, ALT, glucose and ferritin levels, considered as continuous variables). Analyses were carried out with JMP 6.0 statistical analysis software (SAS Institute, Cary, North Carolina, USA).

    Results

    The genotype distribution of all polymorphic alleles was in Hardy–Weinberg equilibrium in both patients and controls. The frequency distributions of the evaluated SNPs were not significantly different between Italian patients with NAFLD and controls (Supplementary table 1 online), but we observed a trend for a higher prevalence of the ENPP1/PC-1 SNP and a lower prevalence of the IRS-1 SNP in patients from the UK than in those from Italy, which was consistent with the reported differences in the prevalence of genetic factors across Europeans populations.27 In the case of ENPP1, genotype frequencies for the patients from the UK with NAFLD were not significantly different from those of a large British control cohort.28 For IRS-1, patients with NAFLD showed a borderline significant decrease in frequency of carriage of the variant compared with a British control group (p = 0.045).29

    Supplemental material

    Effect of the ENPP1/IRS-1 SNPs on metabolic features

    To check whether the genetic variants considered were associated with insulin resistance in patients with NAFLD, we first evaluated the association between the ENPP1 and IRS-1 SNPs with parameters of the metabolic syndrome (table 2). The ENPP1 121Gln allele was significantly associated with higher BMI, waist circumference in both males and females, and with lower high-density lipoprotein (HDL) levels in males. At logistic regression analysis considering sex, BMI and the presence of the ENPP1 SNP as independent variables (ie, variables associated with low HDL levels at univariate analysis), the ENPP1 121Gln allele was independently associated with low HDL levels (<45/55 mg/dl in males/females) (adjusted OR 1.28, 95% CI 1.07 to 1.53, p = 0.007). In contrast, the IRS-1 972Arg allele was associated with lower triglycerides, and nearly associated with a higher prevalence of diabetes. At logistic regression analysis considering age, BMI and the presence of IRS-1 as independent variables (ie, variables associated with diabetes at univariate analysis and the IRS-1 SNP), the 972Arg IRS-1 allele was independently associated with the presence of diabetes (adjusted OR 1.32, 95% CI 1.03 to 1.72, p = 0.02).

    View this table:
    Table 2

    Association of the ENPP1/PC-1 and IRS-1 polymorphisms with metabolic features in 702 patients with NAFLD

    Effect of the ENPP1/IRS-1 SNPs on the severity of liver disease

    The prevalence of NASH, and of fibrosis stage >1 and >2 in the whole series of 702 patients is shown in figure 1. Whereas the prevalence of NASH was not influenced by the ENPP1/IRS-1 genotype status, the prevalence of both fibrosis >1 and fibrosis >2 was significantly higher in patients positive for the ENPP1 121Gln SNP than in patients negative for both the ENPP1/IRS-1 SNPs and than in those positive for the IRS-1 972Arg SNP alone.

    Figure 1
    Figure 1

    Prevalence of non-alcoholic steatohepatitis (NASH), fibrosis stage >1 and fibrosis >2 in 702 biopsied patients with non-alcoholic fatty liver disease (NAFLD) subdivided according to the combined ENPP1 (ectoenzyme nucleotide pyrophosphate phosphodiesterase 1) and IRS-1 (insulin receptor substrate-1) genotypes. p = NS for the frequency distribution of NASH, p = 0.01 for the frequency distribution of fibrosis >1, and p = 0.02 for the frequency distribution of fibrosis >2 among the groups. *p<0.05 vs patients negative for both IRS-1 and ENPP1 single nucleotide polymorphisms (SNPs) (reference group), §p<0.05 vs. patients negative for the IRS-1 SNP.

    The prevalence of fibrosis >1 in patients subdivided according to the geographical origin is shown in table 3. The results in the two major groups of adult patients from Italy and the UK confirmed the overal effect of the ENPP1 121Gln SNP on the severity of liver disease. In Italian patients, the severity of liver disease was significantly more marked in patients positive for the ENPP1 and IRS-1 SNPs than in those positive for the ENPP1 SNP alone, and the presence of the IRS-1 SNP was associated with a higher prevalence of fibrosis >1 in Italian children.

    View this table:
    Table 3

    Effect of combined ENPP1 and IRS-1 genotype on the prevalence of fibrosis stage >1 in patients with NAFLD subdivided according to geographic origin and clinical characteristics

    Independent predictors of fibrosis >1, after adjustment for age, BMI, ALT, glucose and ferritin levels (the clinical variables associated with fibrosis risk at multivariate analysis), and considering the ENPP1 and IRS-1 genotypes as independent variables are shown in table 4. Since the risk of fibrosis for the presence of the ENPP1 SNP appeared to be higher in patients positive than in those negative for the IRS-1 SNP, we reported in table 4 the OR of fibrosis for the ENPP1 SNP considering these two groups separately. The risk of fibrosis >1 was higher in patients carrying both the ENPP1 and the IRS-1 SNPs compared with those who were negative (OR 1.55 95% CI 1.24 and 1.97, p=0.0002), but the effect of the ENPP1 SNP was higher in patients who were also positive for the IRS-1 SNP.

    View this table:
    Table 4

    Clinical and genetic predictors of fibrosis >1 at logistic regression analysis in 702 patients with NAFLD

    Functional effect of ENPP1/IRS-1 SNPs on hepatic insulin signalling

    To check whether the effect of ENPP1/IRS-1 SNPs on liver damage may be mediated by increased hepatic insulin resistance, we evaluated the activation status of the hepatic insulin signalling pathway by measuring pAKT levels in a subgroup of non-diabetic patients with severe obesity who underwent bariatric surgery in Milan. Results are shown in figure 2.

    Figure 2
    Figure 2

    (A) Hepatic AKT activity status (phospho-Thr308 (PT308) and phospho-Ser473 (PS473)) in 24 patients with non-alcoholic steatohepatitis (NASH) subdivided according to histological findings. Data were normalised for β-actin. *p<0.05 vs patients with normal liver (reference group). (B) Hepatic AKT activity status (PT308 and PS473) in 24 patients with NASH subdivided according to the presence or not of the ENPP1 (ectoenzyme nucleotide pyrophosphate phosphodiesterase 1) 121Gln and IRS-1 (insulin receptor substrate-1) 972Arg single nucleotide polymorphisms (SNPs). Data were normalised for β-actin. *p<0.05 vs patients negative for both IRS-1 and ENPP1 SNPs (reference group). White bars, PT308 AKT; black bars, PS473 AKT. (C) Hepatic AKT activity status (PT308 and PS473) as detected by western blotting in 14 representative obese subjects subdivided according to the hepatic pathological findings and the presence of the ENPP1 and IRS-1 SNPs. Total AKT and β-actin levels are shown as a control.

    AKT activity, as detected by the phosphorylation status at position Thr308, was significantly decreased in patients with fatty liver compared with those with normal liver, and to a larger extent in those affected by NASH than in those with pure fatty liver (figure 2A).

    AKT activity, as detected by the phosphorylation status at both position Thr308 and Ser473, was significantly decreased in patients positive for the ENPP1 121Gln or the IRS-1 972Arg alleles compared with patients negative for both SNPs (figure 2B). AKT phosphorylation was ∼30% of that of controls in patients mutated at both Thr308 and Ser473: 27% and 31% in the presence of the IRS-1 and ENPP1 SNPs, respectively, at Thr308, and 21% and 46%, respectively, at Ser473. A representative western blot is shown in figure 2C.

    Discussion

    In this study we evaluated the effect of SNPs of genes involved in the regulation of INSR signalling on liver injury in patients with NAFLD, and found that both the ENPP1 121Gln and IRS-1 972Arg alleles affect insulin resistance and are associated with fibrosis severity.

    Since NAFLD is the hepatic expression of metabolic syndrome, having a strong genetic component, and insulin resistance is associated with disease progression,9 we hypothesised that genetic factors influencing insulin signalling might predispose to liver damage in patients with NAFLD.

    The results indicate that the PC-1/ENPP1 121Gln and IRS-1 972Arg alleles, present in 28.7% and 18.1% of patients, respectively, influence the severity of hepatic fibrosis independently of confounders. Both ENPP1 and IRS-1 interact directly with INSR. ENPP1 is a membrane glycoprotein, which inhibits insulin signalling and when overexpressed causes insulin resistance and related abnormalities. The 121Gln polymorphism is a gain-of-function allele causing stronger interaction with INSR and inhibition of its kinase activity.30 IRS-1 transduces INSR signalling to downstream kinases regulating glucose and lipid metabolism, cell survival and proliferation. The 972Arg polymorphism is a loss-of-function allele characterised by increased association with INSR resulting in decreased autophosphorylation activity,20 and, similarly to what was observed in this study, transgenic mice expressing a mutated human 972Arg allele show increased insulin resistance and decreased β-cell function leading to hyperglycaemia.31 Both genetic variants have consistently been associated with an increased risk of diabetes and metabolic disease.21 ,30 The ENPP1 and IRS-1 SNPs were also associated with insulin resistance-related abnormalities in patients with NAFLD, confirming that their effect may be mediated by insulin resistance. The risk of hyperglycaemia was 32% higher in carriers of the IRS-1 polymorphism, independently of age and BMI, which are well-known risk factors. On the other hand, the ENPP1 121Gln polymorphism seems to determine insulin resistance by a different mechanism, as it was associated with lower HDL values, a sensitive marker of hepatic insulin resistance,32 ,33 and with higher BMI and waist circumference. Interestingly, the ENPP1 121Gln allele has previously been reported to predispose to obesity, possibly by altering appetite control via insulin resistance in the hypothalamus.34

    To provide further support for the effects of these SNPs on hepatic insulin resistance, we were also able to show that they were associated with decreased hepatic pAKT levels, reflecting reduced insulin signalling activity,16 although a limitation of this analysis is that data were available only in a subgroup of obese non-diabetic patients requiring bariatric surgery. Interestingly, the effect of these inherited factors was more marked than that exerted by liver histology, as the presence of fatty liver affected only Thr308 AKT phosphorylation, whereas the presence of ENPP1/IRS-1 SNPs decreased by ∼70% both Thr308 and Ser478 AKT phosphorylation, with a potentially higher effect on insulin signalling.

    Importantly, the effect of the ENPP1 and IRS-1 SNPs on the severity of liver fibrosis was independent of the ethnic background, as it was observed in patients from both Italy and the UK. The higher prevalence of fibrosis >1 in the UK compared with Italy is possibly related to the higher BMI, to the different diet and other as yet unknown genetic factors.

    It is noteworthy that the ENPP1 and IRS-1 SNPs conferred an increased risk of fibrosis in patients with biochemical abnormalities resistant to lifestyle modification, independently of all other clinical predictors identified, including glucose levels. Therefore, the mechanism leading to hepatocellular damage and fibrogenesis involves not only hyperglycaemia, but also decreased cell survival and altered lipogenesis due to an altered INSR/ENPP1/IRS-1 pathway.14 ,15 ,17 Increased metabolic abnormalities and susceptibility to cell death would thus lead to accelerated fibrogenesis in patients carrying these inherited variants.

    Given that in the whole cohort the risk of fibrosis was higher in subjects positive for both the ENPP1 and IRS-1 SNPs compared with those positive for either ENPP1 or IRS-1, a synergic role for the two genetic variants in determining liver damage in patients with NAFLD could be hypothesised. The contemporaneous presence of two defects at the level of INSR may overcome cellular escape mechanisms. In particular, the IRS-1 SNP had no effect when present alone, but increased the risk of fibrosis by almost threefold in the presence of the ENPP1 SNP.

    Although the prevalence of combined heterozygosity for the ENPP1 and IRS-1 SNPs may seem low, from our data and from the prevalence of fatty liver in the general population, it could be roughly estimated that at least 1% of the Western population has NAFLD and carries both the ENPP1 and IRS-1 SNPs, and half of them may already have moderate/severe liver fibrosis. As SNPs are inherited factors, the strong effect of the ENPP1 and IRS-1 SNPs on liver fibrosis suggests that defective insulin signalling may indeed play a causative role in the progression of liver fibrosis in NAFLD, indicating that the association with liver damage is not due to confounders or reverse causation.

    In addition, the data suggest that restoring INSR signalling activity, downregulated in the metabolic syndrome, and also in the liver of patients with NASH16 as shown by AKT phosphorylation status, could be a target of pharmacological treatment in NAFLD. The evaluation of combined ENPP1 and IRS-1 genotypes together with other genes and clinical variables may also play a role in clinical management of these patients if prospective studies confirm our results. This is mainly the case for the increasingly common paediatric NAFLD population, where it is mandatory to identify the most severe cases possibly before severe fibrosis and diabetes develop.

    In conclusion, this study indicates that the ENPP1 121Gln and IRS-1 972Arg SNPs influencing INSR activity predispose to liver damage in Caucasian patients with NAFLD, and suggests that defective INSR-dependent signalling has a causative role in the progression of this liver disease.

    Acknowledgments

    We thank Erika Fatta, Cristina Bertelli and Daniela Bignamini for clinical assistance.

    References

    1. ↵
      1. Browning JD,
      2. Szczepaniak LS,
      3. Dobbins R,
      4. et al
      . Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 2004;40:1387–95.
      OpenUrlCrossRefPubMedWeb of Science
    2. ↵
      1. Bellentani S,
      2. Saccoccio G,
      3. Masutti F,
      4. et al
      . Prevalence of and risk factors for hepatic steatosis in Northern Italy. Ann Intern Med 2000;132:112–17.
      OpenUrlPubMedWeb of Science
    3. ↵
      1. Marchesini G,
      2. Brizi M,
      3. Bianchi G,
      4. et al
      . Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes 2001;50:1844–50.
      OpenUrlAbstract/FREE Full Text
    4. ↵
      1. Ekstedt M,
      2. Franzen LE,
      3. Mathiesen UL,
      4. et al
      . Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology 2006;44:865–73.
      OpenUrlCrossRefPubMedWeb of Science
    5. ↵
      1. Bugianesi E,
      2. Leone N,
      3. Vanni E,
      4. et al
      . Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology 2002;123:134–40.
      OpenUrlCrossRefPubMedWeb of Science
    6. ↵
      1. Day CP
      . From fat to inflammation. Gastroenterology 2006;130:207–10.
      OpenUrlCrossRefPubMedWeb of Science
    7. ↵
      1. Marchesini G,
      2. Bugianesi E,
      3. Forlani G,
      4. et al
      . Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 2003;37:917–23.
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      1. Fracanzani AL,
      2. Valenti L,
      3. Bugianesi E,
      4. et al
      . The risk of severe liver disease in NAFLD with normal aminotransferase levels: a role for insulin resistance and diabetes. Hepatology 2008;48:792–8.
      OpenUrlCrossRefPubMedWeb of Science
    9. ↵
      1. Wilfred de Alwis NM,
      2. Day CP
      . Genetics of alcoholic liver disease and nonalcoholic fatty liver disease. Semin Liver Dis 2007;27:44–54.
      OpenUrlCrossRefPubMedWeb of Science
    10. ↵
      1. Valenti L,
      2. Fracanzani AL,
      3. Dongiovanni P,
      4. et al
      . Tumor necrosis factor alpha promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease. Gastroenterology 2002;122:274–80.
      OpenUrlCrossRefPubMed
    11. ↵
      1. Miele L,
      2. Beale G,
      3. Patman G,
      4. et al
      . The Kruppel-like factor 6 genotype is associated with fibrosis in nonalcoholic fatty liver disease. Gastroenterology 2008;135:282–91.
      OpenUrlCrossRefPubMed
    12. ↵
      1. Valenti L,
      2. Dongiovanni P,
      3. Piperno A,
      4. et al
      . alpha1-Antitrypsin mutations in NAFLD: high prevalence and association with altered iron metabolism but not with liver damage. Hepatology 2006;44:857–64.
      OpenUrlCrossRefPubMedWeb of Science
    13. ↵
      1. Romeo S,
      2. Kozlitina J,
      3. Xing C,
      4. et al
      . Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2008;40:1461–5.
      OpenUrlCrossRefPubMedWeb of Science
    14. ↵
      1. Accili D,
      2. Arden KC
      . FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 2004;117:421–6.
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      1. Valverde AM,
      2. Fabregat I,
      3. Burks DJ,
      4. et al
      . IRS-2 mediates the antiapoptotic effect of insulin in neonatal hepatocytes. Hepatology 2004;40:1285–94.
      OpenUrlCrossRefPubMedWeb of Science
    16. ↵
      1. Valenti L,
      2. Rametta R,
      3. Dongiovanni P,
      4. et al
      . Increased expression and activity of the transcription factor FOXO1 in nonalcoholic steatohepatitis. Diabetes 2008;57:1355–62.
      OpenUrlAbstract/FREE Full Text
    17. ↵
      1. Michael MD,
      2. Kulkarni RN,
      3. Postic C,
      4. et al
      . Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell 2000;6:87–97.
      OpenUrlCrossRefPubMedWeb of Science
    18. ↵
      1. Wieckowska A,
      2. Zein NN,
      3. Yerian LM,
      4. et al
      . In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology 2006;44:27–33.
      OpenUrlCrossRefPubMedWeb of Science
    19. ↵
      1. Grarup N,
      2. Urhammer SA,
      3. Ek J,
      4. et al
      . Studies of the relationship between the ENPP1 K121Q polymorphism and type 2 diabetes, insulin resistance and obesity in 7,333 Danish white subjects. Diabetologia 2006;49:2097–104.
      OpenUrlCrossRefPubMedWeb of Science
    20. ↵
      1. McGettrick AJ,
      2. Feener EP,
      3. Kahn CR
      . Human insulin receptor substrate-1 (IRS-1) polymorphism G972R causes IRS-1 to associate with the insulin receptor and inhibit receptor autophosphorylation. J Biol Chem 2005;280:6441–6.
      OpenUrlAbstract/FREE Full Text
    21. ↵
      1. Jellema A,
      2. Zeegers MP,
      3. Feskens EJ,
      4. et al
      . Gly972Arg variant in the insulin receptor substrate-1 gene and association with type 2 diabetes: a meta-analysis of 27 studies. Diabetologia 2003;46:990–5.
      OpenUrlCrossRefPubMed
    22. ↵
      1. Bedogni G,
      2. Bellentani S,
      3. Miglioli L,
      4. et al
      . The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population. BMC Gastroenterol 2006;6:33.
      OpenUrlCrossRefPubMed
    23. ↵
      1. Kleiner DE,
      2. Brunt EM,
      3. Van Natta M,
      4. et al
      . Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–21.
      OpenUrlCrossRefPubMedWeb of Science
    24. ↵
      1. Sesti G,
      2. Marini MA,
      3. Cardellini M,
      4. et al
      . The Arg972 variant in insulin receptor substrate-1 is associated with an increased risk of secondary failure to sulfonylurea in patients with type 2 diabetes. Diabetes Care 2004;27:1394–8.
      OpenUrlAbstract/FREE Full Text
    25. ↵
      1. Pizzuti A,
      2. Frittitta L,
      3. Argiolas A,
      4. et al
      . A polymorphism (K121Q) of the human glycoprotein PC-1 gene coding region is strongly associated with insulin resistance. Diabetes 1999;48:1881–4.
      OpenUrlAbstract
    26. ↵
      1. von Elm E,
      2. Altman DG,
      3. Egger M,
      4. et al
      . The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet 2007;370:1453–7.
      OpenUrlCrossRefPubMedWeb of Science
    27. ↵
      1. Novembre J,
      2. Johnson T,
      3. Bryc K,
      4. et al
      . Genes mirror geography within Europe. Nature 2008;456:98–101.
      OpenUrlCrossRefPubMedWeb of Science
    28. ↵
      1. Weedon MN,
      2. Shields B,
      3. Hitman G,
      4. et al
      . No evidence of association of ENPP1 variants with type 2 diabetes or obesity in a study of 8,089 U.K. Caucasians. Diabetes 2006;55:3175–9.
      OpenUrlAbstract/FREE Full Text
    29. ↵
      1. Zeggini E,
      2. Parkinson J,
      3. Halford S,
      4. et al
      . Association studies of insulin receptor substrate 1 gene (IRS1) variants in type 2 diabetes samples enriched for family history and early age of onset. Diabetes 2004;53:3319–22.
      OpenUrlAbstract/FREE Full Text
    30. ↵
      1. Prudente S,
      2. Trischitta V
      . The pleiotropic effect of the ENPP1 (PC-1) gene on insulin resistance, obesity, and type 2 diabetes. J Clin Endocrinol Metab 2006;91:4767–8.
      OpenUrlCrossRefPubMed
    31. ↵
      1. Hribal ML,
      2. Tornei F,
      3. Pujol A,
      4. et al
      . Transgenic mice overexpressing human G972R IRS-1 show impaired insulin action and insulin secretion. J Cell Mol Med 2008;12:2096–106.
      OpenUrlCrossRefPubMed
    32. ↵
      1. Suzuki A,
      2. Angulo P,
      3. Lymp J,
      4. et al
      . Chronological development of elevated aminotransferases in a nonalcoholic population. Hepatology 2005;41:64–71.
      OpenUrlCrossRefPubMedWeb of Science
    33. ↵
      1. Tasic I,
      2. Milojkovic M,
      3. Sunder-Plassmann R,
      4. et al
      . The association of PC-1 (ENPP1) K121Q polymorphism with metabolic syndrome in patients with coronary heart disease. Clin Chim Acta 2007;377:237–42.
      OpenUrlCrossRefPubMed
    34. ↵
      1. Goldfine ID,
      2. Maddux BA,
      3. Youngren JF,
      4. et al
      . The role of membrane glycoprotein plasma cell antigen 1/ectonucleotide pyrophosphatase phosphodiesterase 1 in the pathogenesis of insulin resistance and related abnormalities. Endocr Rev 2008;29:62–75.
      OpenUrlCrossRefPubMedWeb of Science

    Footnotes

    • An additional table is published online only at http://gut.bmj.com/content/vol59/issue2

    • PD and LV contributed equally to this work.

    • Funding The work was supported by the following grants: FIRST Università di Milano 2007, 2008 (LV, SF, ALF); Ricerca corrente Ospedale Maggiore Policlinico 2006 and 2008 (LV, SF); and Centro per lo Studio delle Malattie del Fegato e del Metabolismo.

    • Competing interests None.

    • Ethics approval Ospedale Policlinico MaRE IRCCS approved this study.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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