Cholesterol and cholate components of an atherogenic diet induce distinct stages of hepatic inflammatory gene expression

Atherosclerosis in inbred mouse strains has been widely studied by using an atherogenic (Ath) diet containing cholesterol, cholic acid, and fat, but the effect of these components on gene expression has not been systematically examined. We employed DNA microarrays to interrogate gene expression levels in liver of C57BL/6J mice fed the following five diets: mouse chow, the Ath diet, or modified versions of the Ath diet in which either cholesterol, cholate, or fat were omitted. Dietary cholesterol and cholate produced discrete gene expression patterns. Cholesterol was required for induction of genes involved in acute inflammation, including three genes of the serum amyloid A family, three major histocompatibility class II antigen genes, and various cytokine-related genes. In contrast, cholate induced expression of genes involved in extracellular matrix deposition in hepatic fibrosis, including five collagen family members, collagen-interacting proteins, and connective tissue growth factor. The gene expression findings were confirmed by biochemical measurements showing that cholesterol was required for elevation of circulating serum amyloid A, and cholate was required for accumulation of collagen in the liver. The possibility that these gene expression changes are relevant to atherogenesis in C57BL/6J mice was supported by the observation that the closely related, yet atherosclerosis-resistant, C57BL/6ByJ strain was largely resistant to dietary induction of the inflammatory and fibrotic response genes. These results establish that cholesterol and cholate components of the Ath diet have distinct proatherogenic effects on gene expression and suggest a strategy to study the contribution of acute inflammatory response and fibrogenesis independently through dietary manipulation.     

Expression analysis and chromosomal assignment of PRA1 and RILP genes

PRA1 (prenylated Rab acceptor) is a general regulator of Rab proteins, while RILP (Rab interacting lysosomal protein) is a specific effector for Rab7. It has been shown that PRA1 interacts with Rab proteins and with VAMP2. Therefore PRA1 is probably an important factor for membrane traffic, linking together the function of Rab proteins and SNAREs. RILP has a key role in the control of transport to degradative compartments together with Rab7 and probably links Rab7 function to the cytoskeleton. Here we have studied by Northern blot the expression of the two genes in several different human tissues. The 0.8-kb mRNA for human PRA1 is ubiquitously expressed, while the two mRNAs for RILP are differentially expressed. In addition, we have assigned the human PRA1 gene to chromosome 19q13.13-q13.2 and the human RILP gene to chromosome 17p13.3.     

Cloning and characterization of a salt stress-inducible small GTPase gene from the model grass species Lolium temulentum

A gene encoding a small guanosine triphosphate (GTP)-binding protein (smGTP) related to the Rab2 gene family of GTPases was identified during the analysis of a salt stress suppression subtractive hybridization (SSH) expression library from the model grass species Lolium temulentum L. (Darnel ryegrass). The smGTP gene was found to have a low-level constitutive expression and was strongly induced by salt stress in root, crown and leaf tissues. The expression pattern of the smGTP gene was compared against two additional stress genes identified in the SSH expression library, the well-characterized dehydration stress tolerance gene, delta 1-pyrroline-5-carboxylate synthetase (P5CS) encoding for a key enzyme in proline biosynthesis, and the cold response gene COR413. The genes were analyzed for their response to salinity as well as their responses to 7 different forms of abiotic stress in L. temulentum plants. The smGTP gene displayed an expression pattern similar to the P5CS gene, suggesting a role in dehydration stress. In contrast, the COR413 gene was found to be up-regulated in response to all stresses tested and has utility as a general stress marker in grass plants.     

The effect of dietary carbohydrate on genes for fatty acid synthase and inflammatory cytokines in adipose tissues from lean and obese subjects

BACKGROUND: Hepatic de novo lipogenesis (DNL) is markedly stimulated in humans by low-fat diets enriched in simple sugars. However, the dietary responsiveness of the key enzyme controlling DNL in human adipose tissue, fatty acid synthase (FAS), is uncertain. HYPOTHESIS: Adipose tissue mRNA for FAS is increased in lean and obese subjects when hepatic DNL is elevated by a eucaloric, low-fat, high-sugar diet. DESIGN: Twelve lean and seven obese volunteers were given two eucaloric diets (10% vs. 30% fat; 75% vs. 55% carbohydrate; sugar/starch 60/40) each for 2 weeks by a random-order cross-over design. FAS mRNA in abdominal and gluteal adipose tissues was compared to hepatic DNL measured in serum by isotopic and nonisotopic methods. Adipose tissue mRNA for tumor necrosis factor-alpha and IL-6, which are inflammatory cytokines that modulate DNL, was also assayed. RESULTS: The low-fat high-sugar diet induced a 4-fold increase in maximum hepatic DNL (P<.001) but only a 1.3-fold increase in adipose tissue FAS mRNA (P=.029) and no change in cytokine mRNA. There was a borderline significant positive correlation between changes in FAS mRNA and hepatic DNL (P=.039). Compared to lean subjects, obese subjects had lower levels of FAS mRNA and higher levels of cytokine mRNA (P<.001). CONCLUSIONS: The results suggest that key elements of human adipose tissue DNL are less responsive to dietary carbohydrate than is hepatic DNL and may be regulated by diet-independent factors. Irrespective of diet, there is reduced expression of the FAS gene and increased expression of cytokine genes in adipose tissues of obese subjects.     

The response of zinc transporter gene expression of selected tissues in a pig model of subclinical zinc deficiency

This study compared the relative mRNA expression of all mammal zinc (Zn) transporter genes in selected tissues of weaned piglets challenged with short-term subclinical Zn deficiency (SZD). The dietary model involved restrictive feeding (450 g/animal*day⁻¹) of a high-phytate diet (9 g/kg) supplemented with varying amounts of zinc from ZnSO₄*7H₂O ranging from deficient to sufficient supply levels (total diet Zn: 28.1, 33.6, 38.8, 42.7, 47.5, 58.2, 67.8, 88.0 mg Zn/kg). Total RNA preparations comprised jejunal and colonic mucosa as well as hepatic and nephric tissue. Statistical modelling involved broken-line regression (P≤.05). ZIP10 and ZIP12 mRNAs were not detected in any tissue and ZnT3 mRNA was only identified in the kidney. All other genes were expressed in all tissues but only a few gene expression patterns allowed a significant (P<.0001) fitting of broken-line regression models, indicating homeostatic regulation under the present experimental conditions. Interestingly, these genes could be subcategorized by showing significant turnarounds in their response patterns, either at ~40 or ~60 mg Zn/kg diet (P<.0001). In conclusion, the present study showed clear differences in Zn transporter gene expression in response to SZD compared to the present literature on clinical models. We recognized that certain Zn transporter genes were regulated under the present experimental conditions by two distinct homeostatic networks. For the best of our knowledge, this represents the first comprehensive screening of Zn transporter gene expression in a highly translational model to human physiology.     

The mPlrp2 and mClps genes are involved in the hydrolysis of retinyl esters in the mouse liver

Retinyl esters are the major chemical forms of vitamin A stored in the liver, and can be delivered to peripheral tissues for conversion into biologically active forms. The function and regulation of the hepatic genes that are potentially involved in catalyzing the hydrolysis of retinyl esters remain unclear. Here we show that two lipid hydrolytic genes, pancreatic-related protein 2 (mPlrp2) and procolipase (mClps), expressed specifically in the mouse pancreas, are associated with the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illumination deficiency or administration of 5'-AMP elevated the ratio of AdoMet to AdoHcy and induced the expression in the liver of mPlrp2 and mClps, which was blocked by all-trans retinoic acid. Mice fed a vitamin A-free diet exhibited increased activation of hepatic mPlrp2 and mClps expression, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by a methylase inhibitor, methylthioadenosine, markedly decreased plasma retinol levels in these mice. The activated hepatic stellate cell (HSC)-T6 cell line specifically expressed mClps and mPlrp2. Inhibition of mClps gene expressions by short hairpin RNA (shRNA) decreased hydrolysis of retinyl esters in the HSC-T6 cell line. These data suggest that the conditional expression of mPlrp2 and mClps is involved in the hydrolysis of retinyl esters in the mouse liver.     

Effects of Lactobacillus Fermented Soymilk and Soy Yogurt on Hepatic Lipid Accumulation in Rats Fed a Cholesterol-Free Diet

We examined the effects of lactic acid fermented soymilk, in which part of the soymilk was replaced with okara (soy yogurt), on plasma and hepatic lipid profiles in rats fed a cholesterol-free diet. Additionally, we investigated the effects of soy yogurt on hepatic gene expression in rats using DNA microarray analysis. Male Sprague-Dawley rats aged 5 weeks (n=5/group) were fed a control diet (AIN-93) or a test diet in which 20% of the diet was replaced by soy yogurt for 7 weeks. Soy yogurt consumption did not affect body weight or adipose tissue weight as compared with control diet. In the soy yogurt group, the liver weight and hepatic triglyceride content were significantly lower than the control group, and the level of plasma cholesterol was also lower. Furthermore, DNA microarray analysis indicated that soy yogurt ingestion down-regulated the expression of the SREBP-1 gene and enzymes related to lipogenesis in the rat liver, while expression of β-oxidation-related genes was up-regulated. These results suggest that soy yogurt is beneficial in preventing hepatic lipid accumulation in rats.     

橡胶树胶乳4个HbRab基因的克隆和表达分析

克隆了橡胶树胶乳中表达的4个Rab基因的全长cDNA,命名为HbRab5-HbRab8。它们编码22～24kDa的蛋白,均具有小G蛋白家族共有的GTP／GDP结合保守结构域,分别属于植物Rab家族的F、D、A和B亚家族成员。组织表达分析显示,除HbRab69外,其他3个HbRab6基因均在胶乳中表达丰度最高。在胶乳中,伤害处理显著下调HbRab6表达而上调HbRab7表达：乙烯和水杨酸处理下调HbRab6和HbRab8的表达,甲基茉莉酸处理上调HbRab5、HbRab7、HbRab8的表达,细胞分裂素上调HbRab5的表达。本文结果为橡胶树胶乳再生相关Rab基因的筛选与功能阐述奠定了基础。     

Gene expression in atherosclerotic lesion of ApoE deficient mice

BACKGROUND: Atherosclerosis, the major cause of mortality and invalidity in industrialized countries, is a multifactorial disease associated with high plasma cholesterol levels and inflammation in the vessel wall. Many different genes have previously been demonstrated in atherosclerosis, although limited numbers of genes are dealt with in each study. In general, data on dynamic gene expression during disease progress is limited and large-scale evaluation of gene expression patterns during atherogenesis could lead to a better understanding of the key events in the pathogenesis of atherosclerosis. We have therefore applied a mouse gene filter array to analyze gene expression in atherosclerotic ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed atherogenic western diet for 10 or 20 weeks and aortas isolated. C57BL/6 mice on normal chow were used as controls. The mRNAs of 15 animals were pooled and hybridized onto commercially available Clontech mouse gene array filters. RESULTS: The overall gene expression in the ApoE-deficient and control mice correlated well at both time points. Gene expression profiling showed varying patterns including genes up-regulated at 10 or 20 weeks only. At 20 weeks of diet, an increasing number of up-regulated genes were found in ApoE-deficient mice. CONCLUSIONS: The gene expression in atherogenesis is not a linear process with a maximal expression at advanced lesion stage. Instead, several genes demonstrate a dynamic expression pattern with peaks at the intermediate lesions stage. Thus, detailed evaluation of gene expression at several time points should help understanding the development of atherosclerosis and establishment of preventive intervention.     

Dietary beta- and gamma-cyclodextrins stimulation of hepatic metallothionein gene expression in rats

This study investigated whether hepatic metallothionein gene expression is affected by dietary cyclodextrins. Young male Wistar rats were fed a basal diet or cyclodextrin-supplemented (50 g of cyclodextrin per kg diet) diets for 7 d. Copper content in the liver did not show any significant changes among rats fed the basal, beta- and gamma-cyclodextrin diets. There were no differences in liver or serum zinc among groups. Copper content in serum was markedly decreased in rats fed the gamma-cyclodextrin-supplemented diet. Liver metallothionein mRNA levels were significantly elevated in both beta- and gamma-cyclodextrins-fed rats, but not in alpha-cyclodextrin-fed rats. Thus, the increase in hepatic metallothionein mRNA levels might be due to this mechanism except for the contents of copper and zinc in the liver.     

Timed high-fat diet in the evening affects the hepatic circadian clock and PPARα-mediated lipogenic gene expressions in mice

A long-term high-fat diet may result in a fatty liver. However, whether or not high-fat diets affect the hepatic circadian clock is controversial. The objective of this study is to investigate the effects of timed high-fat diet on the hepatic circadian clock and clock-controlled peroxisome proliferator-activated receptor (PPAR) α-mediated lipogenic gene expressions. Mice were orally administered high-fat milk in the evening for 4 weeks. The results showed that some hepatic clock genes, such as Clock, brain-muscle-Arnt-like 1 (Bmal1), Period 2 (Per2), and Cryptochrome 2 (Cry2) exhibited obvious changes in rhythms and/or amplitudes. Alterations in the expression of clock genes, in turn, further altered the circadian rhythm of PPARα expression. Among the PPARα target genes, cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase, low-density lipoprotein receptor, lipoprotein lipase, and diacylglycerol acyltransferase (DGAT) showed marked changes in rhythms and/or amplitudes. In particular, significant changes in the expressions of DGAT and CYP7A1 were observed. The effects of a high-fat diet on the expression of lipogenic genes in the liver were accompanied by increased hepatic cholesterol and triglyceride levels. These results suggest that timed high-fat diets at night could change the hepatic circadian expressions of clock genes Clock, Bmal1, Per2, and Cry2 and subsequently alter the circadian expression of PPARα-mediated lipogenic genes, resulting in hepatic lipid accumulation.     

Rab23 regulates differentiation of ATDC5 chondroprogenitor cells

Insulin treatment of mouse ATDC5 chondroprogenitors induces these cells to differentiate into mature chondrocytes. To identify novel factors that are involved in this process, we carried out mutagenesis of ATDC5 cells through retroviral insertion and isolated two mutant clones incapable of differentiation. Inverse PCR analysis of these clones revealed that the retroviral DNA was inserted into the promoter region of the Rab23 gene, resulting in increased Rab23 expression. To investigate whether an elevated level of Rab23 protein led to inhibition of chondrogenic differentiation, we characterized ATDC5 cells that either overexpress endogenous Rab23 or stably express ectopic Rab23. Our results revealed that up-regulation of Rab23 can indeed inhibit chondrogenic differentiation with a concomitant down-regulation of matrix genes such as type II collagen and aggrecan. In addition, stable small interfering RNA knockdown of Rab23 also resulted in inhibition of chondrogenic differentiation as well as down-regulation of Sox9, a master regulator of chondrogenesis. Interestingly, Sox9 expression has recently been linked to Gli1, and we found that Rab23 knockdown decreased Gli1 expression in chondrocytes. Because the phenotypes of Rab23 mutations in mice and humans include defects in cartilage and bone development, our study suggests that Rab23 is involved in the control of Sox9 expression via Gli1 protein.