Exome-Wide Association Study on Alanine Aminotransferase Identifies Sequence Variants in the GPAM and APOE Associated With Fatty Liver Disease

BACKGROUND & AIMS: Fatty liver disease (FLD) is a growing epidemic that is expected to be the leading cause of end-stage liver disease within the next decade. Both environmental and genetic factors contribute to the susceptibility of FLD. Several genetic variants contributing to FLD have been identified in exome-wide association studies. However, there is still a missing hereditability indicating that other genetic variants are yet to be discovered. METHODS: To find genes involved in FLD, we first examined the association of missense and nonsense variants with alanine amino transferase at an exome-wide level in 425,671 participants from the UK Biobank. We then validated genetic variants with liver fat content in 8930 participants in whom liver fat measurement was available, and replicated 2 genetic variants in 3 independent cohorts comprising 2621 individuals with available liver biopsy. RESULTS: We identified 190 genetic variants independently associated with alanine aminotransferase after correcting for multiple testing with Bonferroni method. The majority of these variants were not previously associated with this trait. Among those associated, there was a striking enrichment of genetic variants influencing lipid metabolism. We identified the variants rs2792751 in GPAM/GPAT1, the gene encoding glycerol-3phosphate acyltransferase, mitochondrial, and rs429358 in APOE, the gene encoding apolipoprotein E, as robustly associated with liver fat content and liver disease after adjusting for multiple testing. Both genes affect lipid metabolism in the liver. CONCLUSIONS: We identified 2 novel genetic variants in GPAM and APOE that are robustly associated with steatosis and liver damage. These findings may help to better elucidate the genetic susceptibility to FLD onset and progression.Peer reviewe

Rare ATG7 genetic variants predispose patients to severe fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disorders and has a strong heritable component. The aim of this study was to identify new loci that contribute to severe NAFLD by examining rare variants

Role of ENPP1 on Adipocyte Maturation

BACKGROUND: It is recognized that the ability of adipose tissue to expand in response to energy excess, i.e. adipocyte maturation, is important in determining systemic abnormalities in glucose and lipid metabolism. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1, also known as PC-1) has been recently reported to be involved in the pathogenesis of insulin resistance and related diseases. However, its role on adipose tissue physiology as a mechanism of systemic insulin resistance is not understood. This study was performed to evaluate whether ENPP1 is regulated during adipogenesis and whether over-expression in adipocytes can affect adipocyte maturation, a potential novel mechanism of ENPP1-related insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: ENPP1 expression was found down-regulated during 3T3-L1 maturation, and over-expression of human ENPP1 in 3T3-L1 (pQCXIP-ENPP1 vector) resulted in adipocyte insulin resistance and in defective adipocyte maturation. Adipocyte maturation was more efficient in mesenchymal embryonal cells from ENPP1 knockout mice than from wild-type. CONCLUSIONS: We identify ENPP1 as a novel mechanism of defective adipocyte maturation. This mechanism could contribute to the pathogenesis of insulin resistance in absence of obesity

PSD3 downregulation confers protection against fatty liver disease

Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD. Employing a candidate gene approach, Mancina et al. identify a genetic variant of the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene that reduces susceptibility to fatty liver disease. Functional studies in vitro and in vivo demonstrate that targeting PSD3 protects against fatty liver disease.Peer reviewe

Role of ENPP1 on Adipocyte Maturation

Background. It is recognized that the ability of adipose tissue to expand in response to energy excess, i.e. adipocyte maturation, is important in determining systemic abnormalities in glucose and lipid metabolism. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1, also known as PC-1) has been recently reported to be involved in the pathogenesis of insulin resistance and related diseases. However, its role on adipose tissue physiology as a mechanism of systemic insulin resistance is not understood. This study was performed to evaluate whether ENPP1 is regulated during adipogenesis and whether over-expression in adipocytes can affect adipocyte maturation, a potential novel mechanism of ENPP1-related insulin resistance. Methodology/Principal Findings. ENPP1 expression was found down-regulated during 3T3-L1 maturation, and over-expression of human ENPP1 in 3T3-L1 (pQCXIP-ENPP1 vector) resulted in adipocyte insulin resistance and in defective adipocyte maturation. Adipocyte maturation was more efficient in mesenchymal embryonal cells from ENPP1 knockout mice than from wild-type. Conclusions. We identify ENPP1 as a novel mechanism of defective adipocyte maturation. This mechanism could contribute to the pathogenesis of insulin resistance in absence of obesity

Human ENPP1 over-expression inhibits DMI-induced adipocyte maturation in 3T3-L1 cells.

<p>Human ENPP1 was over-expressed in 3T3-L1 using retrovirus transfection with CMV promoter. Panel A. ENPP1 protein content in cells containing vector only and in cells over-expressing human ENPP1 K121K or K121Q. ENPP1 was identified at 130 kDa using antibody from Imegex Corp. (San Diego, CA). Panel B. Oil-red-O stained 3T3-L1 cells after induction of adipogenesis. Cells were 100% confluent at the time of assay. Cellular triglyceride content is presented as fold changes from the triglyceride content of cells containing vector. Results are reported as mean and SD from measurements performed three times (8 wells measured in each experiment). Panel C. Cells were plated on 6-well plate and induced to differentiation with DMI. At different days, as indicated, the cell number was counted. Results are reported as mean and SD from measurements repeated three times (3 wells measured in each experiment). Open squares represent the data for the vector 3T3-L1. Open circles represent the data for 3T3-L1 over-expressing human ENPP1 K121K. Dark circles represent the data for 3T3-L1 over-expressing human ENPP1 K121Q. Cell death was measured at day 2 and day 4 of differentiation with DMI. Results are shown as mean and SD of percentage of dead cells relative to total cells. Measurements were performed for number of viable and dead cells in three wells for each cell type. These experiments were repeated 3 times. * p<0.05 for t-test between 3T3-L1 over-expressing human ENPP1 K121K and the vector group. † p<0.05 for t-test between 3T3-L1 over-expressing human ENPP1 K121Q and the vector group.</p

Expression of ENPP1 decreases in the early maturation phase of 3T3-L1 into adipocytes.

<p>3T3-L1 cells were induced to differentiate into adipocytes by desamethasone, 3-iso-butyl-1-methylxanthine and insulin (DMI). The RNA was isolated at different time points, as indicated. Data show real time PCR for expression of ENPP1 and other genes involved in adipocyte maturation. Mean and SD from 3 experiments are presented in arbitrary units after correction for 18S expression. One-way ANOVA p-value for multiple group comparison was <0.05 for each of the gene expression data shown in figure. Pre-adipocyte are shown as dark bars, mature adipocyte are shown as open bars</p

Human ENPP1 K121K and K121Q over-expression induce changes in the expression of genes present of 3T3-L1 induced to maturation.

<p>The expression levels of several adipogenic markers in vector only, in ENPP1 K121K and ENPP1 K121Q stable cell lines 10 days after DMI induction were measured by QPCR and normalized by the level of 18S. Experiments were repeated 3 times. The average values and SD are shown for the vector (open bars) and he transfected cells (dark bars).</p

Gender-dependent association of type 2 diabetes with the vasoactive intestinal peptide receptor 1

Type 2 diabetes is characterized by an inadequate pancreatic beta-cell response to the progressive insulin resistance. Its pathogenesis is complex and has been connected with a state of preclinical chronic inflammation. Vasoactive intestinal peptide (VIP) and its receptors play a relevant role in the homeostasis of insulin secretion as well as in the control of inflammation. In particular, VIP receptor 1 (VPAC1) has been found to be down-modulated during inflammation, and to be associated with several diseases. The objective of this study was to compare the distribution of SNPs mapping in the VIP receptor 1 gene in cases with type 2 diabetes and matched controls. Seven hundred cases with type 2 diabetes (423 males and 277 females) and 830 random controls (419 males and 411 females) were analyzed for the distribution of three common SNPs mapping in the VPAC1 gene. The results show a significantly different genotype distribution of the SNP rs9677 in the 3'-UTR of VPAC1 in female cases with type 2 diabetes compared to gender-matched controls (ptrend = 6 x 10(-4)). The rs9677 CC genotype confers the highest risk (OR: 2.1) and correlates with worse clinical parameters such as higher level of total cholesterol, higher LDL/HDL ratio and a higher HbA1c concentration. The genetic association reported here indicates that VIP/VPAC1 signaling can be a relevant pathway in the pathogenesis of type 2 diabetes in females suggesting that at least some aspects of the genetic predisposition to this disease can be gender-specific. (C) 2011 Elsevier B.V. All rights reserved