Association between the rs3812316 Single Nucleotide Variant of the MLXIPL Gene and Alpha-Linolenic Acid Intake with Triglycerides in Mexican Mestizo Women.

The carbohydrate response element binding protein (ChREBP) is a key transcription factor to understand the gene−diet−nutrient relationship that leads to metabolic diseases. We aimed to analyze the association between the rs17145750 and rs3812316 SNVs (single nucleotide variants) of the MLXIPL gene with dietary, anthropometric, and biochemical variables in Mexican Mestizo subjects. This is a cross-sectional study of 587 individuals. Genotyping was performed by allelic discrimination. In addition, liver and adipose tissue biopsies were obtained from a subgroup of 24 subjects to analyze the expression of the MLXIPL gene. An in silico test of the protein stability and allelic imbalance showed that rs17145750 and rs3812316 showed a high rate of joint heritability in a highly conserved area. The G allele of rs3812316 was associated with lower triglyceride levels (OR = −0.070 ± 0.027, p < 0.011, 95% CI = −0.124 to −0.016), the production of an unstable protein (ΔΔG −0.83 kcal/mol), and probably lower tissue mRNA levels. In addition, we found independent factors that also influence triglyceride levels, such as insulin resistance, HDL-c, and dietary protein intake in women. Our data showed that the association of rs3812316 on triglycerides was only observed in patients with an inadequate alpha-linolenic acid intake (1.97 ± 0.03 vs. 2.11 ± 0.01 log mg/dL, p < 0.001).

Development of an effective and rapid qPCR for identifying human ChREBPα/ß isoforms in hepatic and adipose tissues.

Most quantitative real-time PCR (qPCR) detection methods use two types of chemistries to measure the expression levels of ChREBP isoforms, hydrolysis probes for ChREBPα and SYBR Green for ChREBPß. Hydrolysis probes are not available to determine the ChREBPß isoform. The aim of this study was to develop a qPCR assay based only on hydrolysis probes for both ChREBP isoforms. Liver and adipose tissue biopsies from patients undergoing elective cholecystectomy surgery were used to perform qPCR. To validate this assay, the results were compared with sequencing and High Resolution Melting (HRM) PCR assays. Direct sequencing was used to determine the sequence showing site where ChREBPß presents its specific splicing (1 b exon/2 exon) in order to design the primers and the probe. We developed a qPCR assay to determine the ChREBP isoforms expression based on hydrolysis probes. It assays showed good efficiency (95.50%, on average), high reproducibility, and a strong linear correlation (R2 ≥ 0.99) for tissues tested. HRM analysis confirmed the specificity of the primers and the result of this assay matched (100%) with the outcomes obtained by sequencing and qPCR. Also, we obtained the ChREBPß sequence showing exon 1b spliced to exon 2, bypassing exon 1a, and retaining the remainder of the ChREBPα exons. Based on the use of hydrolysis probes, our method can efficiently identify the expression of both ChREBP isoforms. Thus, the comparability of the qPCR results using a single chemistry (hydrolysis probes) to discriminate between both ChREBP isoforms was possible.

Association of the epsilon 2 allele of APOE gene to hypertriglyceridemia and to early-onset alcoholic cirrhosis.

BACKGROUND: The diverse incidence of alcoholic cirrhosis around the world and the fact that not all alcoholic drinkers develop liver disease indicates that genetic and environmental factors play an important role in the development of liver cirrhosis. Lipids participate in early stages of alcoholic cirrhosis. Therefore variations in the plasma lipid profile due to primary (genetic) or secondary (environmental) dyslipidemia could affect the development of liver disease. The aim of this study was to analyze the lipid profile and apolipoprotein E (APOE) polymorphism in patients with alcoholic liver cirrhosis (AC) and determine the risk associated with genotype polymorphism with the onset of alcoholic cirrhosis. METHODS: In a case and control study, 86 patients with AC divided into hyperlipidemic (H) and non-hyperlipidemic (non-H) groups, and 133 healthy individuals (C) matched by age and sex were studied. Lipid profile and liver function tests were measured by enzymatic methods. The APOE genotypes were identified by PCR-RFLP's. RESULTS: A statistically significant increase of the APOE*2 allele and genotypes 2/2, 2/3, and 2/4 was present in AC patients compared to C group. A hyperlipidemic state characterized by increased levels of triglycerides and apolipoprotein B (APOB) and a decrease of high density lipoprotein-cholesterol (HDL-c) was detected in young-aged patients (31.2 +/- 6.2 years old vs. 46.3 +/- 12.5 years old). In this group, hypertriglyceridemia was closely associated to APOE*2 allele and to an early onset of liver cirrhosis. By contrast, APOE*4 allele was associated with a longer duration of alcohol intake (>20 years) in the non-H group. CONCLUSIONS: This study shows the association of hypertriglyceridemia and APOE allele with the early onset of alcoholic liver cirrhosis, and the interaction between environmental factors, such as duration of alcohol abuse and amount of alcohol intake, and genetic factors (APOE*2 allele) on the hypertriglyceridemic process.

Apolipoprotein AI and apolipoprotein E mRNA expression in peripheral white blood cells from patients with orthotopic liver transplantation.

BACKGROUND: Apolipoprotein AI/apolipoprotein E (apo-AI/apo-E) ratio change and its induction in non-hepatic tissues have been reported during liver development, regeneration, and several pathophysiologic states. The clinical implication of such changes is unclear, but these could reflect recovery and/or severity of liver damage. METHODS AND RESULTS: Using RT-PCR we analysed the mRNA expression of apo-AI and apo-E in peripheral white blood cells (PWBC) of patients with different liver diseases who underwent orthotopic liver transplantation (OLT) and compared its expression with the lipid profile and liver function tests. We found that patients showed higher levels of apo-AI mRNA without detection of apo-E mRNA on PWBC at the preoperative day, compared with healthy volunteers (HV). We found an apo-AI/apo-E mRNA ratio of 2.7 during the anhepatic stage, followed by a decrease to 1.3, 0.95, and 0.55 at days 30, 60, and 90, respectively. At the last time point, the apo-AI/apo-E ratio was similar to HV. At day 3 post-OLT, the lowest levels of high-density lipoprotein (HDL)-cholesterol (17 mg/dl; P<0.05) and the highest levels of aspartate aminotransferase, total bilirubin and alkaline phosphatase (77.5 IU/l, 37.9 g/dl, 177.8 IU/l, respectively; P<0.05) were detected. CONCLUSION: These results indicate that changes of HDL-cholesterol and apo-AI/apo-E mRNA ratio could be a good indicator of liver damage and/or hepatic functional recovery post-OLT.

PI3K is involved in PDGF-beta receptor upregulation post-PDGF-BB treatment in mouse HSC.

Increased expression of PDGF-beta receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF-beta receptor by cathepsin B, thus suggesting that the absence of PDGF-beta receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF-beta receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibitors had no effect. We also showed that receptor reappearance is time and dose dependent, being more delayed in cells treated with 50 ng/ml (48 h) compared with 10 ng/ml (24 h).

TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

Reciprocal modulation of matrix metalloproteinase-13 and type I collagen genes in rat hepatic stellate cells.

Collagen degradation by matrix metalloproteinases is the limiting step in reversing liver fibrosis. Although collagen production in cirrhotic livers is increased, the expression and/or activity of matrix metalloproteinases could be normal, increased in early fibrosis, or decreased during advanced liver cirrhosis. Hepatic stellate cells are the main producers of collagens and matrix metalloproteinases in the liver. Therefore, we sought to investigate whether they simultaneously produce alpha1(I) collagen and matrix metalloproteinase-13 mRNAs. In this communication we show that expression of matrix metalloproteinase-13 mRNA is reciprocally modulated by tumor necrosis factor-alpha and transforming growth factor-beta1. When hepatic stellate cells are co-cultured with hepatocytes, matrix metalloproteinase-13 mRNA is up-regulated and alpha1(I) collagen is down-regulated. Injuring hepatocytes with galactosamine further increased matrix metalloproteinase-13 mRNA production. Confocal microscopy and differential centrifugation of co-cultured cells revealed that matrix metalloproteinase-13 is localized mainly within hepatic stellate cells. Studies performed with various hepatic stellate cell lines revealed that they are heterogeneous regarding expression of matrix metalloproteinase-13. Those with myofibroblastic phenotypes produce more type I collagen whereas those resembling freshly isolated hepatic stellate cells express matrix metalloproteinase-13. Overall, these findings strongly support the notion that alpha1(I) collagen and matrix metalloproteinase-13 mRNAs are reciprocally modulated.