Kazuhito Suruga

Dietary acetic acid reduces serum cholesterol and triacylglycerols in rats fed a cholesterol-rich diet.

To investigate the efficacy of the intake of vinegar for prevention of hyperlipidaemia, we examined the effect of dietary acetic acid, the main component of vinegar, on serum lipid values in rats fed a diet containing 1 % (w/w) cholesterol. Animals were allowed free access to a diet containing no cholesterol, a diet containing 1 % cholesterol without acetic acid, or a diet containing 1 % cholesterol with 0.3 % (w/w) acetic acid for 19 d. Then, they were killed after food deprivation for 7 h. Cholesterol feeding increased serum total cholesterol and triacylglycerol levels. Compared with the cholesterol-fed group, the cholesterol and acetic acid-fed group had significantly lower values for serum total cholesterol and triacylglycerols, liver ATP citrate lyase (ATP-CL) activity, and liver 3-hydroxy-3-methylglutaryl-CoA content as well as liver mRNA levels of sterol regulatory element binding protein-1, ATP-CL and fatty acid synthase (P<0.05). Further, the serum secretin level, liver acyl-CoA oxidase expression, and faecal bile acid content were significantly higher in the cholesterol and acetic acid-fed group than in the cholesterol-fed group (P<0.05). However, acetic acid feeding affected neither the mRNA level nor activity of cholesterol 7alpha-hydroxylase. In conclusion, dietary acetic acid reduced serum total cholesterol and triacylglycerol: first due to the inhibition of lipogenesis in liver; second due to the increment in faecal bile acid excretion in rats fed a diet containing cholesterol.

High-fat and high-cholesterol diet rapidly induces non-alcoholic steatohepatitis with advanced fibrosis in Sprague–Dawley rats

The development of fibrosis is considered an important phase in the progress of non‐alcoholic steatohepatitis (NASH) towards the end stage of liver disease, including cirrhosis. However, few small animal models can display NASH‐associated fibrosis. We aimed to establish a dietary model of NASH with rapid progression to fibrosis using genetically normal rats.

Major intestinal coactivator p300 strongly activates peroxisome proliferator-activated receptor in intestinal cell line, Caco-2

We have previously reported that several genes related to intestinal fatty acid and vitamin A metabolism are coordinately regulated by peroxisome proliferator-activated receptor (PPAR) [Arch. Biochem. Biophys. 389 (2001) 41; Biochim. Biophys. Acta 1531 (2001) 68]. In this study, we demonstrated that PPAR alpha and PPAR delta interacted with endogenous coactivators in intestinal cell line, Caco-2 in a ligand specific manner. We isolated rat cDNA clones encoding the nuclear receptor interaction domains of the two transcriptional coactivators, CREB-binding protein (CBP) and p300. Expression level of CBP mRNA was relatively low in the small intestine, while p300 mRNA was ubiquitously expressed in various tissues including the small intestine in the rat. Southern blot analysis revealed that these coactivators were encoded by different genes. Mammalian two-hybrid assays in Caco-2 cells revealed that p300 interacted with PPAR alpha or PPAR delta in the presence of their specific ligands more efficiently than CBP did. These results suggest that the major intestinal coactivator, p300 strongly interacts with PPAR alpha and PPAR delta.

The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine

In this study, we found that the mRNA level of peroxisome proliferator-activated receptor (PPAR) alpha, but not of PPARdelta, was elevated in the jejunum during the postnatal development of the rat. Moreover, we found that the expressions of PPAR-dependent genes, such as acyl-CoA oxidase, L-FABP, and I-FABP, were also increased during the postnatal development of the small intestine. Electrophoretic mobility shift assay revealed that both the PPARalpha-9-cis-retinoic acid receptor alpha (RXRalpha) heterodimer and the PPARdelta-RXRalpha heterodimer bound to the peroxisome proliferator response element (PPRE) of acyl-CoA oxidase and L-FABP genes. The binding of the PPARalpha-RXRalpha heterodimer to the PPREs of the various genes was enhanced by the addition of PPARalpha, with a concomitant reduction of the binding of PPARdelta-RXRalpha to the PPREs. Furthermore, the binding activity of PPARalpha-RXRalpha, but not PPARdelta-RXRalpha, to the PPREs was enhanced by the addition of a PPAR ligand, WY14,643. The GAL4-PPAR-chimera reporter assay showed that WY14,643 transactivated the reporter gene through action of PPARalpha, but not through PPARdelta, in Caco-2 cells. Furthermore, oral administration of a PPAR ligand, clofibrate, during 3 consecutive days of the weanling period caused a parallel increase in the mRNA levels of these PPAR-dependent genes. These results suggest that acyl-CoA oxidase, L-FABP and the other PPAR-dependent genes in the small intestine may be coordinately modulated during postnatal development by the disproportional expression of PPARalpha over PPARdelta.

Phycocyanin prevents hypertension and low serum adiponectin level in a rat model of metabolic syndrome.

Endothelial dysfunction is associated with hypertension, atherosclerosis, and metabolic syndrome. Phycocyanin is a pigment found in the blue-green algae, Spirulina, which possesses antihypertensive effect. In this study, we hypothesized that phycocyanin derived from Spirulina exerts antihypertensive actions by improving endothelial dysfunction in metabolic syndrome. Spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats were divided into 4 groups then fed a normal diet with or without phycocyanin (2500-, 5000-, or 10,000-mg/kg diet) for 25 weeks. At 34 weeks of age, although systolic blood pressure was not significantly different among groups, phycocyanin-fed groups exhibited a dose-dependent decrease in blood pressure. Serum levels of adiponectin and messenger RNA levels of adiponectin and CCAAT/enhancer-binding protein α in the adipose tissue of rats fed diets containing phycocyanin tended to be higher than those of rats fed a normal diet, but the differences were not statistically significant. Immunohistochemistry analysis showed a significant and positive correlation between aortic endothelial nitric oxide synthase (eNOS) expression levels, a downstream target of the adiponectin receptor, and serum adiponectin levels, although there were no significant differences in eNOS expression among groups. There was also no significant correlation between eNOS expression levels and systolic blood pressure. These results suggest that long-term administration of phycocyanin may ameliorate systemic blood pressure by enhancing eNOS expression in aorta that is stimulated by adiponectin. Phycocyanin may be beneficial for preventing endothelial dysfunction-related diseases in metabolic syndrome.

Effects of miglitol, an α-glucosidase inhibitor, on glycaemic status and histopathological changes in islets in non-obese, non-insulin-dependent diabetic Goto-Kakizaki rats

Miglitol, a 1-deoxynojirimycin derivative, is an alpha-glucosidase inhibitor. In the present study, the effects of acute (single-dose) and chronic (8-week) oral administration of miglitol in Goto-Kakizaki (GK) rats, an animal model of type 2 diabetes, were investigated. Dose-dependent decreases in incremental blood glucose concentrations integrated over a period of 2 h (deltaAUC0-2 h) for values of blood glucose after sucrose-loading in miglitol-treated GK rats were observed following an acute oral administration of miglitol (1, 3 or 10 mg/kg body weight). At 10 mg/kg, the deltaAUC0-2 h of blood glucose was decreased by 45 % compared with the control group. Following the oral administration of miglitol in a dietary mixture (10 mg, 20 mg or 40 mg miglitol/100 g control diet) for 8 weeks, the ratio of HbA1c at 8 weeks compared with 0 weeks in GK rats treated with 40 mg miglitol/100 g control diet miglitol was significantly decreased compared with control GK rats without changes in body weight. In oral glucose tolerance testing, miglitol caused a slight decrease in the deltaAUC0-2 h of plasma glucose concentration. In addition, miglitol treatment slightly inhibited the reduction in beta-cell mass, and lessened the irregular contours and fibrosis of the islets in GK rats. These results indicate that miglitol ameliorates the hyperglycaemic state of GK rats and the impaired function of the pancreatic islets, as well as preventing the degeneration of islets in GK rats.

Dietary fatty acids are possible key determinants of cellular retinol-binding protein II gene expression

We previously found that dietary unsaturated fatty acids increase cellular retinol-binding protein type II (CRBP II) mRNA and its protein levels in rat jejunum. To obtain insight into mechanisms for its gene induction, we investigated the effect of depletion of dietary fat on CRBP II mRNA levels and we further examined whether dietary retinol is necessary for dietary fat-induced CRBP II gene expression. Feeding the fat-free diet, which contained a sufficient amount of vitamin A, repressed CRBP II mRNA accumulation by 50% within 1 day, and this low level was sustained over the next 9 days. Parallel to the decreased CRBP II mRNA level, the peroxisomal proliferator-activated receptor-alpha (PPAR-alpha) mRNA level in rat jejunum was decreased by long-term (7 days) feeding of an isocaloric low-fat diet compared with the control. Oral administration of corn oil in the animals fed vitamin A-free diet elicited approximately threefold accumulation of CRBP II mRNA within 6 h. However, the administration of 9-cis-retinoic acid brought about no accumulation of CRBP II mRNA. Even when rats were vitamin A-deficient, oral administration of corn oil, but not 9-cis-retinoic acid, caused an increase in jejunal CRBP II mRNA level. These results suggest that CRBP II gene expression in rat jejunum may be regulated predominantly by dietary fatty acids but little by dietary retinoids.

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells

Summary.Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.

Peroxisome proliferator enhances gene expression of cellular retinol-binding protein, type II in Caco-2 cells

Both the mRNA and protein of cellular retinol-binding protein, type two (CRBP(II)) are induced in rat intestine by high fat (corn oil) diet (Biochim. Biophys. Acta 1200, 34-40, 1994) as well as by dietary unsaturated long-chain fatty acids (J. Nutr. 125, 2039-2044, 1995). To gain an insight into the mechanism for this induction, we investigated whether CRBP(II) gene was activated by exposure of the human intestinal cell line, Caco-2 to a peroxisome proliferator (clofibric acid) and/or 9-cis retinoic acid. Northern blot hybridization revealed that Caco-2 cells endogenously expressed the mRNAs of peroxisome proliferator-activated receptor alpha (PPARalpha) and retinoid X receptor alpha (RXRalpha). The expression of the genes encoding CRBP(II), PPARalpha, and RXRalpha increased progressively during differentiation of Caco-2 cells. The cells exposed to 100 microM clofibric acid exhibited 70% greater CRBP(II) mRNA and the exposure of the cells to 100 microM clofibric acid in combination with 100 microM 9-cis retinoic acid exhibited 130% greater CRBP(II) mRNA level, indicating that the effect of the combination of them was additive. Neither PPARalpha mRNA nor RXRalpha mRNA level was enhanced by clofibric acid. In conclusion, our data suggested that the CRBP(II) gene expression may be enhanced by an activation of PPARalpha-RXRalpha heterodimer through some putative metabolite(s) formed via fatty acid-related metabolic pathway in the clofibrc acid-treated cells.

Cloning of chick cellular retinol-binding protein, type II and comparison to that of some mammals: Expression of the gene at different developmental stages, and possible involvement of RXRs and PPAR☆

We cloned chick cellular retinol-binding protein, type two (CRBP II) cDNA and compared it with those of some mammals. The deduced amino acid sequence showed that chick CRBP II was one amino acid greater in size than those of mammals, and the nucleotide sequence of chick CRBP II shared 72%-75% similarity with those of mammals. RNA blot hybridization analysis showed that CRBP II transcript of 0.7 kb was first detected in the duodenum of day-18 embryonic chick, and exhibited a rapid increase during 24 hr around the hatching. Northern blot hybridization also revealed that the transcripts of two types of retinoid X receptors (RXR alpha and RXR gamma) and peroxisome proliferator-activated receptor (PPAR) were expressed in the chick duodenum at hatching. The organ culture of day 16 embryonic chick duodenum showed that the addition of 9-cis retinoic acid in the medium caused a significant increase in CRBP II mRNA levels. In addition, arachidonic acid, from which putative ligands for PPAR were supposed to be generated, was accumulated around hatching in the duodenum. The results may suggest that the abrupt increase of the CRBP II gene expression in the chick duodenum around hatching may be related with RXRs and/or PPAR.