Dietary freshwater clam (Corbicula fluminea) extract suppresses accumulation of hepatic lipids and increases in serum cholesterol and aminotransferase activities induced by dietary chloretone in rats.

We investigated the ameliorative effect of freshwater clam extract (FCE) on fatty liver, hypercholesterolemia, and liver injury in rats exposed to chloretone. Furthermore, we examined the effects of major FCE components (fat and protein fractions) to determine the active components in FCE. Chloretone increased serum aminotransferase activities and led to hepatic lipid accumulation. Serum aminotransferase activities and hepatic lipid content were lower in rats fed total FCE or fat/protein fractions of FCE. Expression of fatty acid synthase and fatty acid desaturase genes was upregulated by chloretone. Total FCE and fat/protein fractions of FCE suppressed the increase in gene expression involved in fatty acid synthesis. Serum cholesterol levels increased twofold upon chloretone exposure. Total FCE or fat/protein fractions of FCE showed hypocholesterolemic effects in rats with hypercholesterolemia induced by chloretone. These suggest that FCE contains at least two active components against fatty liver, hypercholesterolemia, and liver injury in rats exposed to chloretone.

Freshwater Clam Extract Ameliorates Triglyceride and Cholesterol Metabolism through the Expression of Genes Involved in Hepatic Lipogenesis and Cholesterol Degradation in Rats.

The freshwater clam (Corbicula spp.) is a popular edible bivalve and has been used as a folk remedy for liver disease in Asia. As a Chinese traditional medicine, it is said that freshwater clam ameliorates alcoholic intoxication and cholestasis. In this study, to estimate the practical benefit of freshwater clam extract (FCE), we compared the effects of FCE and soy protein isolate (SPI) on triglyceride and cholesterol metabolism in rats. FCE and SPI lowered serum cholesterol, and FCE tended to reduce serum triglycerides. FCE enhanced fecal sterol excretion and hepatic mRNA levels of CYP7A1 and ABCG5 more substantially than SPI; however, both diets reduced hepatic cholesterol. Both of the diets similarly suppressed liver lipids improved Δ9-desaturated fatty acid profile, and FCE was associated with a reduction in FAS and SCD1 mRNA levels. Hepatic transcriptome analysis revealed that inhibition of lipogenesis-related gene expression may contribute to downregulation of hepatic triglycerides by FCE. FCE would have better potential benefits for preventing metabolic disorders, through greater improvement of metabolism of triglycerides and cholesterol, likely through a mechanism similar to SPI.

Lipid components prepared from a freshwater Clam (Corbicula fluminea) extract ameliorate hypercholesterolaemia in rats fed high-cholesterol diet.

To explore the hypocholesterolaemic components in the fat fraction of freshwater clam extract (FCE), we further fractionated the fat fraction by silica gel column chromatography into nine fat subfractions. In the present study, we used exogenous hypercholesterolaemic rats induced by feeding a high-cholesterol diet; the doses of the added fat subfractions were equivalent to those in 30% FCE. Two (FF1, FF2) out of the nine fat subfractions strongly reduced serum cholesterol levels in the rats fed a high-cholesterol diet. Both FF1 and FF2 up-regulated the hepatic gene expression of cholesterol 7α-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis. Thin-layer chromatography showed that FF1 primarily contained sphingolipids, while FF2 mainly contained triacylglycerols and sterol esters. These results indicate that fractions containing sphingolipids, triacylglycerols, and sterol esters are possibly responsible for the hypocholesterolaemic action in a novel manner through the up-regulation of the hepatic biosynthesis of bile acids.

Spherical cell shape of FLC-4 cell, a human hepatoma cell, enhances hepatocyte-specific function and suppresses tumor phenotype through the integration of mRNA-microRNA interaction.

The induction mechanism of HNF-4α by spherical cell shape in human hepatoma cells, FLC-4, was investigated. To get insight into the induction mechanism of HNF-4α in three-dimensional FLC-4 cells, mRNA microarray analysis was performed. The gene expression related to drug metabolism and nuclear receptors, such as LXRα, was elevated in spherical FLC-4 cells. We found the first time that the expressions of genes related to malignancy of hepatoma cells, such as HIF-1α, c-Myc and VEGFC, were downregulated by spherical cell shape. Network analysis revealed that HNF-4α would elicit both the enhancement of hepatocyte-specific gene expression and suppression of malignancy. Since HNF-4α gene expression was known to be regulated by microRNA, we inferred that spherical cell shape would induce HNF-4α gene expression through microRNA. To investigate the possibility of such a mechanism, mRNA-microRNA interactions were examined using microRNA microarray and bioinformatics analysis. The level of miR-24, a microRNA targeting HNF-4α, was reduced in spherical FLC-4 cells. On the other hand, spherical cell shape-induced miR-194 and miR-320c would directly downregulate SLC7A5 and E2F1 gene expression, respectively, which are both related to malignancy. Our study suggested that spherical cell shape would induce HNF-4α gene expression and consequent enhancement hepatocyte-specific functions. Spherical cell shape itself would suppress malignancy in FLC-4 cells through microRNA, such as miR-194 and miR-320c.

Real-time monitoring in three-dimensional hepatocytes reveals that insulin acts as a synchronizer for liver clock.

Resetting the peripheral clock and understanding the integration between the circadian rhythm and metabolic pathways are fundamental questions. To test whether insulin acts as a synchronizer for the hepatic clock by cell-autonomous mechanisms, the phase-resetting capabilities of insulin were investigated in cultured hepatic cells. We provide evidence that three-dimensional (3D) cell culture conditions that preserve the differentiated state of primary hepatocytes sustained the robustness of the molecular clock, while this robustness rapidly dampened under classical monolayer cell culture conditions. Herein, we established a 3D cell culture system coupled with a real-time luciferase reporter, and demonstrated that insulin directly regulates the phase entrainment of hepatocyte circadian oscillators. We found that insulin-deficient diabetic rats had a pronounced phase advance in their hepatic clock. Subsequently, a single administration of insulin induced phase-dependent bi-directional phase shifts in diabetic rat livers. Our results clearly demonstrate that insulin is a liver clock synchronizer.