Plasma lipoprotein profile from nonalcoholic steatohepatitis model rats.

We recently revealed that increases in particle sizes of very-low-density lipoproteins (VLDL) are highly correlated with the progression of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), and VLDL particle size may be a minimally invasive indicator of these hepatic disorders. Methionine and choline-deficient (MCD) diet fed animals are usually used as a NASH model; however, the application of this minimally invasive biomarker in MCD diet fed animals remains unclear. In the present study, we measured the levels of liver disease markers and plasma lipoprotein profiles in MCD diet fed rats, and compared them with those of normal diet fed rats. Assessing lipoprotein profiles showed marked increases in VLDL particle sizes in MCD diet fed rats with pathologically and biochemically NASH-like features.

Lipoprotein profile and lipid metabolism of PXB-cells®, human primary hepatocytes from liver-humanized mice: proposal of novel in vitro system for screening anti-lipidemic drugs.

We investigated lipid metabolism in PXB-cells, which are human primary hepatocytes isolated from liver-humanized mice, and HepG2 and HuH-7 human hepatoma cell lines. Lipoprotein levels were higher in PXB-cells than in the 2 other cell lines, and PXB-cells mainly released triglycerides and cholesterol as very low density lipoprotein (VLDL), similar to actual liver tissue, whereas the major lipoprotein released from the 2 hepatoma cell lines was LDL. RT-PCR analysis demonstrated that the gene expression levels of apolipoprotein B100 (ApoB100), the apolipoprotein of VLDL/LDL, were similar in PXB-cells and HepG2 cells, while the overexpression of ApoC2, ApoC3, and ApoE, which are components of VLDL, but not LDL, was observed in PXBcells. A protein immunoassay revealed that ApoB100 levels secreted from PXB-cells and HuH-7 cells were similar; however, ApoC3 levels were higher in PXB-cells than in the two other cell lines. We also examined the anti-lipidemic activities of fenofibrate using this assay system. Fenofibrate suppressed lipoprotein production from PXB-cells in a dose-dependent manner mainly by activating the ß-oxidation pathway. These results suggest that PXB-cells produce high levels of lipoproteins and are suitable for screening anti-lipidemic agents.

Lipoprotein profiles of hepatic cell lines at various stages of differentiation.

We studied the lipoprotein profiles of human hepatic cells at various stages of differentiation. The production of three major classes of lipoproteins, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), was detected in three well-differentiated human hepatoma cell lines and primary human hepatocytes; however, these lipoproteins were not detected in the culture medium in which undifferentiated hepatoma cell lines were grown. Reverse transcription polymerase chain reaction analysis demonstrated that the expression levels of apolipoprotein A1 (ApoA1), ApoB100, and microsomal triglyceride transfer protein (MTP) were markedly lower in the undifferentiated hepatoma cell lines than in the well-differentiated hepatoma cell lines and primary hepatocytes. These results indicate that apolipoprotein synthesis, and triglyceride-transport by MTP might be rate-limiting steps in lipoprotein production in mature hepatic cells.

Oral choline tolerance test as a novel noninvasive method for predicting nonalcoholic steatohepatitis.

BACKGROUND: Although therapeutic intervention for nonalcoholic steatohepatitis (NASH) at an early stage is important owing to the progressive nature of the disease, diagnosis using noninvasive methods remains difficult. We previously demonstrated NASH specific impairment of choline metabolism and the use of fasting plasma free choline (fCh) levels for NASH diagnosis. Here, we investigated the utility of an oral choline tolerance test (OCTT), based on disordered choline metabolism, as a novel noninvasive method for NASH diagnosis. METHODS: Sixty-five patients with biopsy proven nonalcoholic fatty liver disease (NAFLD) and 17 healthy controls were enrolled. Blood samples were obtained from all subjects five times during the OCTT (before and 1, 2, 3, and 4 h after oral loading with 260 mg choline). RESULTS: Four-hour fCh levels after oral loading choline were markedly increased in NASH patients, compared with non-NASH subjects. For detecting NASH, compared with non-NASH subjects, the area under the curve for 4-h fCh levels was 0.829 on receiver operating characteristic (ROC) analysis. The cut-off level for NASH diagnosis was ≥0.16 mg/dL, and the sensitivity, specificity, positive predictive value, and negative predictive value were 80.1, 82.6, 78.4, and 84.4 %, respectively. Moreover, 4-h fCh levels were significantly associated with the disease activity based on NAFLD activity score in patients with NAFLD. CONCLUSIONS: Four-hour fCh levels obtained by an OCTT reflect a NASH specific disorder of choline metabolism, suggesting that the OCTT is a novel and useful noninvasive method for diagnosing NASH at an early stage with sufficient accuracy for clinical practice.

An in vitro assay system for antihyperlipidemic agents by evaluating lipoprotein profiles from human intestinal epithelium-like cells.

We developed an in vitro screening system for antihyperlipidemic activity by measuring lipoprotein profiles secreted from human intestinal epithelium-like cells from the colon cancer cell line, Caco-2. Sodium (Na) butyrate at 5 mM differentiated Caco-2 cells into intestinal epithelium-like cells and numerous microvilli on the apical side of cells were observed under transmission electron microscopy. Real-time RT-PCR analysis revealed that Na butyrate stimulated expression levels of intestinal differentiation markers in Caco-2 cells in a dose-dependent manner and 5 mM Na butyrate up-regulated intestinal alkaline phosphatase, sucrase-isomaltase complex, and microsomal triglyceride transfer protein by 8.1-, 1.9-, and 2.1-fold that of non-treated cells, respectively. Lipoprotein secretions from differentiated Caco-2 cells were promoted by lysophosphatidyl choline and Na oleate, which are a stimulator of lipoprotein secretion and a substrate of triglycerides, respectively. We examined the effects of Pluronic L-81, a lipoprotein secretion inhibitor, on lipoprotein profiles of differentiated Caco-2 cells. Pluronic L-81 at 1.0 µg/ml inhibited TG contents in lipoprotein fractions from cells by 25.6 % and secretion was completely suppressed by the agent at 10 µg/ml.

Amino acid sequence of Egyptian goose egg-white lysozyme and effects of amino acid substitution on the enzymatic activity.

The amino acid sequence of Egyptian goose lysozyme (EGL) from egg-white and its enzymatic properties were analyzed. The established sequence had the highest similarity to wood duck lysozyme (WDL) with five amino acid substitutions, and had eighteen substitutions difference from hen egg-white lysozyme (HEL). Tyr34 and Gly37 were found at subsites E and F of the active site when compared with HEL. The experimental time-course characteristics of EGL against the N-acetylglucosamine pentamer substrate, (GlcNAc)(5), revealed higher production of (GlcNAc)(4) and lower production of (GlcNAc)(2) when compared with HEL. The saccharide-binding ability of subsites A-C in EGL was also found to be weaker than in HEL. An analysis of the enzymatic reactions of five mutants in respect of positions 34, 37 and 71 in HEL indicated the time-course characteristics of EGL to be caused by the combination of three substitutions (F34Y, N37G and G71R) between HEL and EGL. A computer simulation of the EGL-catalyzed reaction suggested that the time-course characteristics of EGL resulted from the difference in the binding free energy for subsites A, B, E and F and the rate constant of transglycosylation between EGL and HEL.

In vitro screening for antihyperlipidemic activities in foodstuffs by evaluating lipoprotein profiles secreted from human hepatoma cells.

We screened the antihyperlipidemic effects of seven edible plants by evaluation of the triglyceride (TG) and cholesterol profiles secreted from HepG2 cells. We found that the water- and ethanol-extracts of Brasenia schreberi at 100 µg/ml exhibited strong inhibitory activities against TG and cholesterol secretions from HepG2 cells stimulated with sodium oleate. Real-time RT-PCR analysis demonstrated that ethanol extract of B. schreberi (BSET) attenuated the expression of the sterol regulatory element binding protein-1c and -2, fatty acid synthase and HMG CoA synthase-1 genes, which are involved in lipid synthesis in hepatocyte/hepatoma cells. Furthermore, we studied the action of BSET on adipose tissue accumulation and serum parameters in mice fed a high-fat diet (HFD). BSET suppressed mesenteric and epididymal adipose tissue accumulation and normalized serum TG and glucose, but not cholesterol levels in HFD-fed mice.

Normative values and correlates of carotid artery intima-media thickness and carotid atherosclerosis in Andean-Hispanics: The Prevencion Study.

OBJECTIVES: Carotid intima-media thickness (cIMT) is an independent predictor of cardiovascular risk. Furthermore, ethnicity and gender-specific normative data are required to assess cIMT, which are not available for Andean-Hispanics. In addition, data regarding correlates of subclinical atherosclerosis in ethnic population are needed. METHODS: We studied 1448 adults enrolled in a population-based study in Peru. cIMT and carotid plaque were measured with high-resolution ultrasonography. A healthy reference sample (n=472) with no cardiovascular disease, normal weight and normal metabolic parameters was selected to establish normative cIMT values. Correlates of abnormal cIMT and carotid plaque were assessed in the entire population. RESULTS: In the reference sample, 95th-percentile cIMT values were both age and gender-dependent. In stepwise regression, selected predictors of increasing cIMT were: older age, impaired fasting glucose, diabetes mellitus, higher systolic blood pressure, higher LDL-cholesterol, smoking and male gender. Predictors of carotid plaque included older age, male gender, higher systolic blood pressure, lower diastolic blood pressure and higher LDL-cholesterol. HDL-cholesterol and C-reactive protein were not associated with cIMT or carotid plaque. The lack of association with HDL-cholesterol was confirmed using high performance liquid chromatography. CONCLUSIONS: We present ethnic-specific cut-offs for abnormal cIMT applicable to Andean-Hispanics and correlates of subclinical atherosclerosis in this population. Pending longitudinal studies, our data supports several risk associations seen in other populations and can be used to identify Andean-Hispanics at increased risk for atherosclerotic cardiovascular disease. The lack of association between HDL-C and cIMT or carotid plaque in this population requires further investigation.

HPLC analysis of lipoproteins in culture medium of hepatoma cells: an in vitro system for screening antihyperlipidemic drugs.

We isolated a HepG2-derived sub-clone (HepG2-Lipo), which possessed an increased lipoprotein synthesizing ability. HepG2-Lipo cells could secrete triglycerides (TG) and cholesterol at rates 9.4- and 6-fold higher, respectively, when compared to HepG2 cells. Real-time RT-PCR analysis revealed that the expression levels of sterol regulatory element-binding protein-1c and -2 were 2.9- and 1.5-fold higher than in HepG2 cells. Furthermore, two apolipoprotein (apo) genes (apoA-1 and apoB-100) in HepG2-Lipo cells were expressed at 2.8- and 1.9-fold higher levels when compared to those in parental cells. We examined the effects of three antihyperlipidemic agents on the lipoprotein profiles of HepG2-Lipo cells. Simvastatin at 5 microM selectively suppressed cholesterol secretion from HepG2-Lipo cells, and 500 microM fenofibrate inhibited both TG and cholesterol secretion from the cells.

Histidine-114 at subsites E and F can explain the characteristic enzymatic activity of guinea hen egg-white lysozyme.

The courses of the reaction catalyzed by guinea hen egg-white lysozyme (GHL), in which Asn113 and Arg114 at subsites E and F in hen egg-white lysozyme (HEL) are replaced by Lys and His, respectively, was studied with the substrate N-acetylglucosamine pentamer, (GlcNAc)5. Although GHL was found to retain the main-chain folding similar to HEL as judged from CD spectroscopy, the courses of GHL showed increased production of (GlcNAc)4 and reduced production of (GlcNAc)2 when compared with HEL. To identify critical residue(s) involved in the alteration in the courses of GHL, two mutant enzymes as to subsites E and F in HEL, N113K and R114H, were prepared by site-directed mutagenesis. Kinetic analysis of these mutants revealed that the mutation of Asn113 to Lys had little effect on the courses of HEL, while the Arg114 to His mutation completely reproduced the courses of GHL, demonstrating that His114 in GHL is the key residue responsible for the characteristic courses of GHL. Computer simulation of the reaction courses of the R114H mutant revealed that this substitution decreased not only the binding free energies for subsites E and F, but also the rate constant of transglycosylation. The Arg residue at position 114 may play an important role in the transglycosylation activity of HEL.

The amino acid sequence of satyr tragopan lysozyme and its activity.

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).

Amino acid residues in subsites e and f responsible for the characteristic enzymatic activity of duck egg-white lysozyme.

We analyzed the enzymatic properties of duck egg-white lysozyme II (DEL), which differs from hen egg-white lysozyme (HEL) in nineteen amino acid substitutions. A substrate binding study showed that DEL binds to the substrate analog at subsites A-C in the same manner as HEL. However, the experimental time-courses of DEL against the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed remarkably enhanced production of (GlcNAc)(2) and reduced production of (GlcNAc)(1) as compared to in the case of HEL. Computer simulation of the DEL-catalyzed reaction suggested that the amino acid substitutions at subsites E and F (Phe34 to Tyr and Asn37 to Ser) caused the great alteration in the time-courses of DEL. Subsequently, the enzymatic reactions of mutants, in which Phe34 and Asn37 in HEL were converted to Tyr and Ser, respectively, were characterized. The time-courses of the F34Y mutant exhibited profiles similar to those of HEL. In contrast, the characteristics of the N37S mutant were different from those of HEL and rather similar to those of DEL; the order of the amounts of (GlcNAc)(1) and (GlcNAc)(2) was reversed in comparison with in the case of HEL. Enhanced production of (GlcNAc)(2) was also observed for the mutant protein, F34Y/N37S, with two substitutions. These results indicated that the substitution of Asn37 with Ser can account, at least in part, for the characteristic time-courses of DEL. Moreover, replacement of Asn37 with Ser reduced the rate constant of transglycosylation. The substitution of the Asn37 residue may affect the transglycosylation activity of HEL.