Junichiro Takahashi

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.

Plasma free choline is a novel non-invasive biomarker for early-stage non-alcoholic steatohepatitis: A multi-center validation study.

Aim:  Choline is a dietary component that is crucial for normal cellular function. Choline is predominantly absorbed from the small intestine and completely metabolized in the liver. We recently demonstrated that free choline (fCh) levels in blood reflect the level of phosphatidylcholine synthesis in the liver and is correlated with the onset of non‐alcoholic steatohepatitis (NASH). Our aim here was to validate the utility of this biomarker for NASH diagnosis.

Hepatic triglyceride lipase plays an essential role in changing the lipid metabolism in genotype 1b hepatitis C virus replicon cells and hepatitis C patients

Recently, several studies have shown the existence of associations between lipoprotein profiles and hepatitis C virus (HCV), although only a limited amount of information is available about the mechanisms underlying the changes in the lipoprotein profiles associated with HCV. In this study, we investigated the association between lipoprotein profile, classified according to the particle size, and lipoprotein metabolism.

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.

Advantages of assessing lipoprotein profiles in hepatic cell differentiation

Recently, mature hepatocytes have attracted a great deal of attention in drug metabolism or toxicities instead of liver tissue. The human hepatoma cell line, HepG2, was found to chemically differentiate into mature hepatocyte-like cells by sodium (Na) butyrate, and some of the properties of mature hepatocytes were induced by their differentiation (Wang et al. 1998; Fukuda et al. 2005); however, changes in lipid metabolism and lipoprotein production in the cells, which are important functions of hepatocytes, remain unclear. Lipoproteins are complexes of cholesterol, triglycerides (TG), phospholipids, and apolipoproteins (Apo) and are responsible for transporting lipids in the bloodstream. Serum lipoproteins have been classified into four major classes, chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins, and high density lipoproteins (HDL) by their density and size. The liver is a major organ for lipid metabolism and mainly secretes serum lipids as three lipoproteins, VLDL, LDL, and HDL; therefore, many antihyperlipidemic agents target improvements in lipid metabolism in the liver. We previously developed an in vitro assay system to assess lipoprotein profiles from human hepatoma cells (Itoh et al. 2009; Takahashi et al. 2011). Herein, we studied the relationships between HepG2 cell differentiation and its lipoprotein production, and whether measuring lipoprotein profiles in the cultured medium was a useful tool for evaluating the differentiation stages of hepatic cells. Briefly, we studied the effects of Na butyrate at concentrations of 1–5 mM on gene expression levels in HepG2 cells. These cells (5×10 cells) were seeded and precultured in Dulbecco’s modified Eagle’s medium (DMEM, 1 ml) containing 10% fetal calf serum (FCS) in 24-well plates with or without Na butyrate for 4 d. The isolation of total RNA and real-time RT-PCR were performed as previously described (Itoh et al. 2009). The relative expression level of each mRNA was normalized by the amount of GAPDH mRNA. The results were summarized in Table 1 (the primer sequences used in this study are shown in Table S1). Na butyrate at 1–2 mM markedly induced two liver markers, albumin and transferrin. CYP1A1 and CYP3A4, chytocrome P-450 members, were elevated at Na butyrate concentrations over 2 mM. Microsomal triglyceride transfer protein (MTTP), localized to the lumen of the endoplasmic reticulum in the liver and intestine, has been identified as a factor necessary for the assembly of ApoB. LDL receptors are widely distributed in various tissues and were shown to be highly expressed in the liver. Na butyrate at 1–2 mM increased the mRNA levels of these proteins. These results demonstrated that Na butyrate induced hepatic genes in HepG2 cells, and 2 mM Na butyrate in particular efficiently differentiated HepG2 cells to mature hepatic-like cells. We simultaneously investigated the effects of Na butyrate on the gene expression levels of lipoprotein components (Apos, TG, and cholesterol). The genes of ApoA1 and ApoB-100, components for HDL and VLDL/LDL, respectively, were elevated by 1–2 mM Na butyrate. Sterol regulatory-element binding protein (SREBP)-1c and fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) are a lipogenic transcriptional factor and enzymes, respectively. SREBP-2, HMG-CoA synthase-1 (HMGCS-1), and squalene synthase are essential for cholesterogenesis. Na butyrate at Electronic supplementary material The online version of this article (doi:10.1007/s11626-013-9639-8) contains supplementary material, which is available to authorized users. J. Takahashi : F. Kimura :M. Miura :Y. Iwama :G. Toshima Skylight Biotech Inc, 100-4 Sunada, Iijima, Akita 011-0911, Japan