Superiority of dietary safflower oil over olive oil in lowering serum cholesterol and increasing hepatic mRnas for the LDL receptor and cholesterol 7alpha-hydroxylase in exogenously hypercholesterolemic (exHC) rats

The exogenously hypercholesterolemic (ExHC) rat is a strain segregated from SD rats with a high response to dietary cholesterol. To understand the underlying mechanism(s) for this hypercholesterolemia, the interactive effects of dietary fatty acid and the susceptibility of rats to dietary cholesterol on the serum cholesterol concentration and hepatic mRNA abundance of the low-density lipoprotein (LDL) receptor, cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) and 3-hydroxyl-3methylglutaryl (HMG) CoA reductase were examined. Both strains were fed on a diet supplemented with 10% each of olive, safflower or coconut oil with or without the addition of 1% cholesterol for one week. The ExHC rats fed on olive, safflower and coconut oil in combination with cholesterol respectively resulted in a 3.5-, 2.0- and 2.1-fold higher serum cholesterol concentration than that in the animals fed on the corresponding dietary fats without any supplementation of cholesterol (p < 0.01 by dietary cholesterol or type of fat). The dietary cholesterol dependent-elevation of serum cholesterol in the SD rats was less than 1.5-fold (p<0.01) and there was no dietary fat effect. The ExHC rats fed on the safflower oil-containing diet supplemented with cholesterol resulted in a higher mRNA abundance of the LDL receptor and 7alpha-hydroxylase than in the corresponding fat-fed rats without cholesterol (p<0.05). There was no dietary cholesterol-dependent change of mRNA abundance in either strain fed on olive or coconut oil, except for a decreased abundance of HMG CoA reductase mRNA in the olive oil-fed ExHC rats and coconut oil-fed Sprague-Dawley (SD) rats (p<0.05). These results indicate that the hepatic mRNA abundance of the LDL receptor and of 7alpha-hydroxylase depended on the dietary combination of cholesterol and a fatty acid and suggest that a linoleic acid-rich diet may alleviate exogenous hypercholesterolemia by activating the process involved in the hepatic uptake and biliary excretion of serum cholesterol.     

Exogenously Hypercholesterolemic Rats, Compared with Their Progenitor Sprague-Dawley Rats, Have Altered mRNAs for Cholesterol 7α-Hydroxylase and Low-Density-Lipoprotein Receptor and Activities of Cholesterol 7α-Hydroxylase and Acyl-CoA:Cholesterol Acyltransferase in the Liver in Response to Dietary Cholesterol

Exogenously hypercholesterolemic (ExHC) rats promptly increase serum cholesterol concentration in response to dietary cholesterol. To examine underlying mechanism(s) for this susceptibility, responses of mRNAs for cholesterol metabolism-related proteins and their activities in the liver to dietary cholesterol were compared between ExHC rats and their progenitor Sprague-Dawley rats. ExHC rats slightly decreased the abundance of low-density-lipoprotein (LDL) receptor mRNA in response to dietary cholesterol, although the amount of LDL receptor was not influenced. The abundance of cholesterol 7α-hydroxylase mRNA and the enzyme activity in response to dietary cholesterol were greater in ExHC rats, but the fecal excretion of bile acid was comparable between the strain. Dietary cholesterol-dependent elevation of acyl-CoA:cholesterol acyltransferase activity was greater in ExHC rats. The concentration of liver triacylglycerols was markedly lower in ExHC rats. These results suggest that ExHC rats may increase serum cholesterol by increasing hepatic secretion of cholesteryl ester-rich particles.     

Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats.

Exogenous hypercholesterolemic (ExHC) rats, that develop hypercholesterolemia for exogenous cholesterol, are an established strain Isolated from Sprague-Dawley (SD) rats by Imai and Matsumura ((1973) Atherosclerosis, 18, 59-64). The present study was carried out to clarify the cause of hyperresponsivity in ExHC rats to dietary cholesterol. As early as one day after feeding a high cholesterol diet (1%) serum cholesterol level was doubled in ExHC rats, while the level of hepatic cholesterol was two-thirds of SD rats. The elevation of serum cholesterol was mainly attributed to the d less than 1.006 g/ml fractions. Cholesterol feeding increased fecal bile acid excretion in both strains, but to a more greater extent in SD rats. Absorption of dietary cholesterol and synthesis of cholesterol in vivo were similar between the strains. The uptake of beta-very-low-density-lipoproteins (beta-VLDL) in vivo and the primary cultured hepatocytes was lower in ExHC rats, when a high-cholesterol diet was fed. Even without feeding of a high-cholesterol diet, preincubation with cholesterol-rich lipoproteins caused a lower association and degradation of beta-VLDL by the hepatocytes from ExHC rats. Incubation of hepatocytes with cholesterol-rich lipoproteins did not affect the secretion of [14C]cholesterol into the density less than 1.006 g/ml fraction, but suppressed the secretion into the medium density greater than 1.006 g/ml fractions. These results suggest that ExHC rats, as compared to SD rats, are defective of hepatic uptake and processing cholesterol to bile acids.     

Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats

The exogenously hypercholesterolemic (ExHC) rat is an established strain that exhibits a polygenic syndrome of hypercholesterolemia after feeding on a cholesterol-containing diet, and the extent of this differs between male and female rats in the strain. The present study was performed to determine the genetic background of diet-induced hypercholesterolemia in ExHC rats. We used quantitative trait locus (QTL) analyses of the F2 progeny derived from ExHC and Brown-Norway rats. Rats were fed a diet containing 1% cholesterol, and a genome-wide scan was then performed. Significant QTLs for serum total cholesterol levels were revealed on chromosomes 5 and 14 in the vicinity of markers D5Rat95 and D14Rat43, having maximum logarithm of the odds scores of 6.0 and 5.8, respectively. A suggestive QTL for the trait was also detected on chromosome 3 at D3Rat140. In particular, the QTL on chromosome 5 was specific for female rats. These loci were novel QTLs for post-dietary serum total cholesterol levels. In addition, cross-mating analysis in F1 generations suggested that the responsiveness to dietary cholesterol in ExHC rats is partly attributable to X-linked inheritance. Identifying such genetic factors may be useful in predicting the risks associated with diet-induced hypercholesterolemia in humans.     

Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol

Early responses of cholesterol metabolism to dietary cholesterol were compared between exogenous hypercholesterolemic (ExHC) and Sprague-Dawley rats. Both strains had a similar radioactivity of [¹⁴C]cholesterol in the serum half a day after the oral administration, but thereafter the radioactivity disappeared slowly in ExHC rats. ExHC rats promptly altered in response to the dietary cholesterol, activities of cholesterol 7α-hydroxylase and cholesterol synthesis in the liver and fecal excretion of bile acids derived from [¹⁴C]cholesterol administered orally. Lymphatic transport for 24 hr of [¹⁴C]cholesterol was similar between the strains. Triton administration resulted in a marked accumulation of cholesterol in serum d > 1.006 g/ml lipoproteins in ExHC rats; in addition, the formation of cholesteryl esters from [¹⁴C]oleic acid intravenously infused was greater in ExHC rats. These results indicate that ExHC rats increase serum cholesterol in response to exogenous cholesterol by decreasing the liver uptake and enhancing the secretion in the liver.     

Screening for ribosomal-based false positives following prokaryotic mRNA differential display

Differential display (DD) and the closely related RNA arbitrarily primed PCR (RAP-PCR) have become the molecular tools of choice for identifying and isolating differentially expressed genes in both eukaryotic and prokaryotic systems. However, one of the current drawbacks of both techniques is the high number of false positives generated. In prokaryotic applications, the many false positive typically generated by DD are subsequently identified as rRNAs because of their greater abundance compared to mRNAs. To circumvent this problem, full-length 16S and 23S rDNA probes, derived from Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1, respectively, were used as a prescreening approach to discriminate between those bands, which appear to be differentially expressed mRNAs, but in fact are rRNAs, following prokaryotic mRNA DD.     

Effect of PPAR-delta agonist on the expression of visfatin, adiponectin, and resistin in rat adipose tissue and 3T3-L1 adipocytes

It has been recently reported that activation of PPAR-delta, by specific agonists or genetic manipulation, alleviates dyslipidemia, hyperglycemia, and insulin resistance in animal models of obesity and type 2 diabetes. The purpose of the present study was to determine whether the PPAR-delta agonist has a direct effect on adipokines in visceral adipose tissue of rats and in cultured adipocytes. We examined the expression of visfatin, adiponectin, and resistin mRNA in visceral adipose tissue of Wistar rats fed a high-fat diet and 3T3-L1 adipocytes treated with PPAR-delta agonist (L-165041). Body weight and biochemical measurements were performed. Rats fed a high-fat diet showed a greater increase in body weight than those fed a standard diet (P<0.05), and treatment with L-165041 (10 mg/kg/day) significantly decreased weight gain (P<0.05). The concentration of total cholesterol was lower, and HDL cholesterol was higher in L-165041-treated rats (P<0.05). In the visceral adipose tissue of L-165041-treated rats, visfatin and adiponectin mRNA levels significantly increased compared to those of the untreated rats (P<0.05). However, the expression of resistin decreased in the L-165041-treated rats. Furthermore, in cultured 3T3-L1 adipocytes, the level of visfatin and adiponectin mRNA was up-regulated in response to L-165041 treatment for nine days. By contrast, resistin mRNA levels were down-regulated by L-165041 treatment. The present study provides a novel evidence to suggest that the PPAR-delta agonist has regulatory effects on a variety of adipokines, and these effects might explain some of their metabolic function.     

Normal Distribution of Body Weight Gain in Male Sprague‐Dawley Rats Fed a High‐Energy Diet

Objective: To investigate the effect of a high‐energy (HE) diet on caloric intake, body weight, and related parameters in outbred male Sprague‐Dawley (SD) rats. Research Methods and Procedures: Twenty‐eight SD rats were fed either chow (C) for 19 weeks or HE diet for 14 weeks and then C for 5 weeks. Blood hormones and metabolites were assayed, and expression of uncoupling protein‐1 and hypothalamic energy‐balance‐related genes were determined by Northern blotting and in situ hybridization, respectively. Results: HE rats gained body weight more rapidly than C animals with a range of weight gains, but there was no evidence that weight gain was bimodally distributed. Caloric intake was transiently elevated after introduction of the HE diet. Transfer of HE rats back to C resulted in a drop in caloric intake, but a stable body weight. In terminal analysis, two of four dissected adipose tissue depots were heavier in rats that had previously been fed HE diet. Blood leptin, insulin, glucose, and nonesterified fatty acids were not different between the groups. Uncoupling protein‐1 mRNA was elevated in interscapular brown adipose tissue from HE rats. There was a trend for agouti‐related peptide mRNA in the hypothalamic arcuate nucleus to be higher in HE rats. Discussion: Contrary to other studies of the SD rat on HE diet, body weight and other measured parameters were normally distributed. There was no segregation into two distinct populations on the basis of susceptibility to diet‐induced obesity. This characteristic may be dependent on the breeding colony from which animals were sourced.     

Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice, We combined the RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice. We believe that the findings described here will help to elucidate the molecular mechanism(s) underlying the developmental processes of rice leaf.     

RNA fingerprinting of specific plant cell types: Adaptation to plants and optimization of RNA arbitrarily primed PCR (RAP-PCR)

Differential gene expression contributes to cell differentiation and underlies plant responses to hormonal and environmental factors. Most methods available to identify differentially expressed genes in plants are biased towards moderately or strongly expressed genes. RNA arbitrarily primed polymerase chain reaction (RAP-PCR) produces populations of amplicons from reverse transcribed RNA in a process similar to differential display, but with a higher degree of reproducibility and sensitivity, thus enabling the identification of low abundance mRNA. A detailed RAP-PCR protocol allowing the rapid identification of differentially expressed genes in scarce plant cells, such as stomatal guard cells, is presented here. In addition, a fast and reliable method for the semi-quantitative confirmation of gene expression patterns is described.     

Feeding unsaponifiable compounds from rice bran oil does not alter hepatic mRNA abundance for cholesterol metabolism-related proteins in hypercholesterolemic rats

The hypocholesterolemic effect of rice bran oil (RBO) is defined in human and animal experiments which indicate the presence of active component(s) in the unsaponifiable fraction, but the detailed mechanism is not known yet. Exogenously hypercholesterolemic (ExHC) rats were fed for 2 weeks on a 0.5% cholesterol diet supplemented with 10% each of RBO, RBO-simulated oil (RBOSO) in its fatty acid composition, or RBOSO plus 0.25% unsaponifiable compounds (UC) from RBO. Rats fed RBO or the UC resulted in lowing serum and liver cholesterol concentration and preventing reduction of high density lipoproteinic-cholesterol. Dietary RBO or the UC led to an elevation of fecal neutral sterol excretion, but no significant change in fecal bile acid excretion or in hepatic abundance of mRNAs for 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol-7alpha-hydroxylase, and low density lipoprotein receptor. Besides, serum and liver alpha-tocopherol concentrations were lowered in RBO or the UC-fed rats. These results show that the UC in RBO leads to a decreased serum cholesterol concentration by interrupting the absorption of intestinal hydrophobic compounds rather than by modifying cholesterol metabolism in the liver.     

The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display

In many applications, an understanding of differentially expressed genes in different tissues, or owing to an applied stimulus is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR [RAP-PCR] and differential, display [DDR-PCR] has shown, that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR a technique has recently been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application. We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR) is a reliable and useful modification of available differential expression methods.     

Hepatic expression of apolipoprotein A-I gene in rats is upregulated by monounsaturated fatty acid diet

The effect of the degree of dietary fat saturation on the hepatic expression of apolipoprotein A-I mRNA was studied in male rats. Animals were maintained for two months on a high fat diet (40% w/w) containing 0.1% cholesterol. Two groups of control animals received either chow diet or chow plus 0.1% cholesterol, while experimental groups received their fat supplement as coconut, corn or olive oil respectively. Dietary cholesterol did not affect apolipoprotein A-I mRNA levels as compared to control animals. Corn oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA than those receiving cholesterol, or coconut oil plus cholesterol. Olive oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA when compared to all other dietary groups. Our data indicate that monounsaturated fatty acids supplied as olive oil play a major role in regulating the hepatic expression of apolipoprotein A-I in male rats.     

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.     

Hypocholesterolemia of Rhizoma Coptidis alkaloids is related to the bile acid by up-regulated CYP7A1 in hyperlipidemic rats

This study is to investigate the cholesterol-lowering effect and the new mode of action of coptis alkaloids on high lipid diet-induced hyperlipidemic rats. Coptis alkaloids extract (CAE) was prepared by alcohol extraction from Rhizoma Coptidis that have been quality-controlled according to the protocol. The cholesterol-lowering effect of CAE was evaluated on SD rats fed with high-lipid diet. Serum level of lipid, Bile acid and cholesterol in the liver and feces of the rats were measured using colorimetric assay kit. RT-PCR and Western blot were used to analyze the mRNA and protein expression of cholesterol metabolism-related genes including cholesterol 7α-hydroxylase (CYP7A1), peroxisome proliferator-activated receptor-alpha (PPARα) and farnesoid X receptor (FXR) in the livers of the rats. A HPLC analysis was used to assess the activity of CYP7A1. The results showed that CAE reduced the levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C). CYP7A1 gene expression and its activity was up-regulated dose-dependently accompanying with the increased level of bile acid and the reduced cholesterol level in the livers of the CAE treated hyperlipidemic rats. Meanwhile, the mRNA expression of PPARα was also up-regulated in dose-dependent way accompanying the down-modulation of the FXR mRNA expression in the livers of the CAE treated hyperlipidemic rats. The results indicate that the cholesterol-lowering effect of coptis alkaloid extract is at least partly attributed to its promoting the cholesterol conversion into bile acids by up-regulating the gene expression of CYP7A1 and thus increasing its activity in the liver of the hyperlipidemic rats, which might related to the positive regulation of PPARα and the negative modulation of FXR.     

Subtractive differential display: a modified differential display technique for isolating differentially expressed genes

Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need to be sequenced to establish the gene identity.     

Induction of pancreatic trypsin by dietary amino acids in rats: four trypsinogen isozymes and cholecystokinin messenger RNA

We previously demonstrated that feeding a diet containing a high level of amino acid mixture simulating casein (AA) induced an increase in pancreatic protease activities in rats. In the present study, this effect of dietary AA was further characterized with three separate experiments. These experiments (1) examined periodic changes in pancreatic and small intestinal trypsin activities after switching from a 20% (a normal nitrogen level) AA diet to a 60% AA (a high nitrogen level) diet; (2) measured the abundance of mRNA for four trypsinogen isozymes and for intestinal cholecystokinin (CCK) and secretin in rats fed 20% and 60% AA diets for 10 days compared with rats fed 20% and 60% casein diets; and (3) measured the abundance of mRNA for four trypsinogen isozymes after chronic administration of CCK. Trypsin activities were gradually increased in both the pancreas and the small intestinal lumen and reached maximum at 5 days after the switch to the 60% AA diet (Exp. 1). This result is evidence that the increase in the protease activity in the pancreas is due to enhancement of pancreatic trypsin production. In experiment 2, pancreatic trypsinogen isozymes I, II, III, and IV mRNA abundance were evaluated by the Northern blotting method using cDNA probes specific for each isozyme mRNA. Abundance of trypsinogen mRNA without trypsinogen I tended to increase in the rats fed the 60% casein diet but tended to decrease in the rats fed the 60% AA diet compared with the respective normal nitrogen level diet groups without significant difference. CCK mRNA abundance in the jejunal mucosa increased as a result of feeding the 60% casein diet, but not the 60% AA diet. Subcutaneous CCK injections (3.5 nmole/kg body weight/day, twice daily, at 8:30 am and 7:30 pm) for 10 days resulted in increased pancreatic trypsin activity, whereas the changes in mRNA of the four trypsinogen isozymes was similar between the 20% and 60% casein groups but differed between the 20% and 60% AA groups (Exp. 3). These results suggest that CCK is not involved in the induction of pancreatic trypsin that occurs with feeding of a high AA diet and that the mechanism of protease induction by dietary AA is different from that in the case of dietary protein.