10 research outputs found

    Serum ferritin level is associated with liver fibrosis and incident liver-related outcomes independent of <i>HFE</i> genotype in the general population

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    Hyperferritinemia reflects iron accumulation in the body and has been associated with metabolic disturbances and alcohol use, and is also a common finding in individuals diagnosed with liver disease. The major genetic regulator of iron metabolism is the HFE gene. The aim of this this study was to investigate the association between serum ferritin and liver fibrosis using the enhanced liver fibrosis (ELF) test, and the association between ferritin and liver-related outcomes in a Finnish population-based cohort of 6194 individuals (45% male, mean [± standard deviation] age, 52.9 ± 14.9 years; body mass index 26.9 ± 4.7 kg/m2). The effects of HFE variants on these associations were also evaluated. Serum ferritin levels were significantly associated with liver fibrosis, as estimated by enhanced liver fibrosis (ELF) test in weighted linear regression analysis. Serum ferritin was significantly associated with both all liver-related outcomes (n = 92) and severe liver-related outcomes (n = 54) in weighted Cox regression analysis (hazard ratio [HR] per 1 SD, 1.11 [95% confidence interval (CI) 1.02–1.21]; p = 0.012 and HR 1.11 [95% CI 1.02–1.21]; p = 0.013, respectively). However, there was association neither between HFE risk variants and ELF test nor between HFE risk variants and liver-related outcomes. Serum ferritin levels were associated with liver fibrosis and incident liver disease, independent of HFE genotype in the general population. Furthermore, data demonstrated that metabolic disturbances and alcohol use were major risk factors for hyperferritinemia.</p

    Table1_Serum MMP-8 and TIMP-1 concentrations in a population-based cohort: effects of age, gender, and health status.docx

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    BackgroundMatrix-degrading proteinases and their regulators, such as matrix metalloproteinase 8 (MMP-8) and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), may contribute to various pathological events. Elevated MMP-8 concentrations have been associated with e.g., cardiovascular diseases and periodontitis. However, there is little knowledge on physiological concentrations of these molecules in serum, or the effect of demographic or lifestyle factors on their levels.Design and methodsWe investigated the effect of various demographic characteristics and behavioral habits, such as aging, sex, smoking, and BMI, on serum concentrations of MMP-8 and TIMP-1. We used the FINRISK97 cohort (n = 8,446), which has comprehensive information on demographic and lifestyle factors, clinical data, laboratory measurements, and register data available. Further, we investigated the concentrations of MMP-8, TIMP-1, and the MMP-8/TIMP-1 ratio in different age groups of healthy and diseased participants. T-test was used to compare log-transformed mean levels in different groups and linear regression was used to evaluate the association between MMP-8 and TIMP-1 and selected diseases and background variables.ResultsMMP-8 levels decreased with increasing age in the whole population and for women, while TIMP-1 concentration increased slightly with age for the whole population and both genders separately (p for linear trend ConclusionThe association of serum MMP-8 and TIMP-1 concentrations with cardiometabolic risk is frequently investigated. MMP-8 levels decrease significantly with age and fasting time. In addition, sex, smoking, and obesity are associated with both MMP-8 and TIMP-1 concentrations. These factors should be carefully considered in epidemiological studies on serum MMP-8 and TIMP-1.</p

    The ORP8 ligand-binding domain binds cholesterol.

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    <p><b>A</b> The purified His<sub>6</sub>-ORP8 ORD preparation, Coomassie blue stained SDS-PAGE gel. The two major bands (indicated, ORP8) represent the ORP8 ORD fusion protein. <b>B</b> The ability of His<sub>6</sub>-ORP8 ORD to extract [<sup>3</sup>H]cholesterol from unilamellar PC∶cholesterol (99∶1 mol%) vesicles was assayed. Purified GST was used as a negative control. Each assay contained 9.9 nmol PC and 100 pmol cholesterol; The protein amounts used (25–100 pmol) are indicated at the bottom. The data represents % of total DPM extracted from the vesicles, a mean ± s.e.m. (GST, n = 4; His<sub>6</sub>-ORP8 ORD, n = 5).</p

    ORP8 overexpression reduces plasma and hepatic lipids in mice.

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    <p><b>A</b> Western analysis of adenovirus (Ad)-mediated ORP8 overexpression in mouse liver. Liver total protein (20 µg/lane) from mice at day 5 after infection with AdGFP and AdORP8 was Western blotted with ORP8 antibody. <b>B</b> Plasma Cholesterol (Chol), choline phospholipids (PL), and triglycerides (TG) measured from animals at 5 days after infection with AdGFP or AdORP8. <b>C</b> Hepatic lipid levels of the mice at day 5. <b>D</b> Mouse liver nuclear (1st and 3rd panel) or total (2nd and 4th panel) protein fractions (40 µg/lane) at day 5 after infection were Western blotted with antibodies against SREBP-1 or SREBP-2. nSREBP, nuclear SREBP; pSREBP, precursor SBEBP. (E) Analysis of SREBP target gene mRNAs (identified at the bottom) in the liver of mice transduced with AdGFP (open bars) or AdORP8 (closed bars) by qPCR. The data represents mean ± s.e.m. (*p<0.05; **p<0.01; n = 5; t-test).</p

    ORP8 interacts with Nup62.

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    <p><b>A</b> Bimolecular fluorescence complementation (BiFC) analysis of ORP8 interaction with Nup62. HuH7 cells were cotransformed for 24 h with plasmids encoding the fusion proteins Nup62/pVn-C1 and ORP8/pVc-C1 or ORP8pVc-N1 (indicated on the left) for 24 h, followed by 48 h incubation with 10 µg/ml cycloheximide. ER-DsRed2 was contransfected as a transfection control and ER marker. BiFC (GFP channel) and DsRed fluorescence were imaged (identified at the top). Bar, 10 µm. <b>B</b> Lysate of untransfected HuH7 cells was immunoprecipitated with anti-ORP8 (identified at the top) or an irrelevant control IgG, followed by Western blot analysis with anti-Nup62 (top panel) or anti-ORP8 (bottom panel). H, IgG heavy chain.</p

    Primers used for mRNA quantification by real-time RT-PCR.

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    <p>The abbreviations are: LDL-R, low-density lipoprotein receptor; HMG-CR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; HMG-CS, 3-hydroxy-3-methylglutaryl coenzyme A synthetase; FAS, fatty acid synthetase; ACS, acetyl-CoA synthetase; SCD-1, stearoyl-CoA desaturase 1. The prefix m indicates mouse and h human sequence.</p

    ORP8 co-localizes with Nup62 at the nuclear envelope: Confocal microscopy analysis.

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    <p>HuH7 cells were transfected with ORP8, ORP1L, ORP3, or ORP10 cDNA for 24 h using Lipofectamine 2000, followed by processing for confocal immunofluorescence microscopy double staining with anti-Nup62 (green) and anti-ORP (red) antibodies. <b>A–C</b> Nup62 and ORP8 localization in transfected Huh7 cells. Co-localization of ORP8 and Nup62 at the nuclear envelope is indicated with arrows in the channel merge panel. No Nup62 colocalization was observed with ORP1L (<b>D</b>), ORP3 (<b>E</b>), or ORP10 (<b>F</b>). Bars, 10 µm. <b>G</b> Analysis of ORP8, 1L, 3, or 10 (identified in the panels) colocalization with Nup62 at the nuclear envelope in representative cells. Fluorescence intensity (on an arbitrary scale) of the nuclear circumference at the Nup62 (green) and ORP (red) channels was quantified by using the Leica LCS software.</p

    Identification of the interacting domains in Nup62 and ORP8.

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    <p><b>A</b> Schematic presentation of the Nup62 prey constructs used in yeast two-hybrid assays. The numbers indicate amino acid positions. <b>B</b> Interaction of the Nup62 constructs and ORP8m (lacking the C-terminal trans-membrane segment). Yeast colonies grown on SD/2- plates (left), SD/4- plates (middle) and x-gal assay (right) are shown. <b>C</b> Schematic presentation of the ORP8 bait constructs used. <b>D</b> Interaction of the ORP8 constructs with Nup62 in the yeast two-hybrid assay. Yeast colonies grown on SD/2- plates (top), SD/4- plates (middle) and x-gal assay (bottom) are shown. pGADT7 is the empty prey vector and pGBKT7 the empty bait vector.</p

    Nup62 is involved in reduction of nuclear SREBPs by excess ORP8.

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    <p><b>A</b> HuH7 cells were transfected with empty vector plasmid (Mock), <i>ORP8</i> cDNA (ORP8), non-targeting siRNA (siNT) or siNup62 as indicated, followed by preparation of total protein specimens and nuclear fractions, and Western blot analysis thereof with antibodies against SREBP-1 and SREBP-2. Nuclear SREBPs (nSREBPs) decreased in cells overexpressing ORP8 (the top panels), while no change in nSREBPs was seen in cells transfected with siNup62 (the two bottom panels). <b>B</b> HuH7 cells were transfected with combinations of siRNAs and plasmids as indicated at the top. Nuclear fractions Western blotted for SREBP-1 and SREBP-2, and total protein specimens (40 µg/lane) blotted for Nup62, ORP8, and β-actin are shown at the bottom. <b>C,D</b> Quantification of the relative levels of nSREBP-1 and nSREBP-2 after the indicated combined transfections. <b>E</b> Effect of the combined transfections on the mRNA levels of SREBP target genes (identified at the bottom); qPCR analysis. The results represent mean ± s.e.m. (n = 4, *p<0.05, **p<0.01, t-test; difference to values of the siNT and Mock-transfected control, which was set at 1).</p

    Silencing and overexpression of ORP8 have opposite effects on SREBP target gene expression in HuH7 cells.

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    <p><b>A</b> Western analysis of HuH7 cells subjected to ORP8 silencing and overexpression. Stable expression of scrambled control shRNA (shNT) or shORP8, or overexpression by plasmid transfection for 36 h using Neon™ electroporation (ORP8; Mock = transfection with empty vector). <b>B</b> Quantification of selected SREBP target gene mRNAs (identified at the bottom) in cells expressing shNT or shORP8. <b>C</b> Quantification of selected mRNAs (identified at the bottom) in cells transfected with the empty vector (Mock) or <i>ORP8</i> cDNA (ORP8). <b>D</b> Cholesterol biosynthesis as measured by [<sup>3</sup>H]acetic acid incorporation (30 min pulse, 90 min chase), in cells expressing shNT or shORP8. The result is given as % of the biosynthesis in shNT-expressing cells. <b>E</b> Cholesterol biosynthesis in cells transfected with the plain vector (Mock) or <i>ORP8</i> cDNA (ORP8) for 36 h. The data represents mean ± s.e.m. (*p<0.05, **p<0.01, n = 4–6; t-test).</p
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