Comparison of Receptors and Enzymes Regulating Cholesterol Levels in Liver between SHR/NDmcr-cp Rats and Normotensive Wistar Kyoto Rats at Ten Weeks of Age

Abstract

Spontaneously hypertensive rat (SHR)/NDmcr-cp (cp/cp) rat (SHR-cp) is a congenic SHR rat with nonsense mutation introduced into the leptin receptor.1) Metabolic syndrome, characterized by the concomitant presence of obesity, hypertension, hyperlipidemia and diabetes, develops spontaneously in the SHR-cp, 2-4) along with age-related histologically evident glomerular injury and tubulointerstitial damage, including mesangial activation, podocyte injury, and inflammatory cell infiltration in the tubulointerstitium. 5) SHR-cp was reported to show hypercholesteremia and hyperglyceridemia, as compared with lean littermates (SHR-ln) that have hypertension.2) The serum total cholesterol level in SHR-cp at 18 weeks of age is higher than that of Wistar Kyoto rat (WKY), 6) but that in SHR-cp at 10 weeks of age is the same.7) It remains unclear whether there are differences in the system regulating serum cholesterol levels between SHRcp and WKY at 10 weeks of age. Thus, we compared cholesterol levels in serum and liver between SHR-cp and WKY. Furthermore, mRNA levels of receptors or enzymes involved in uptake from serum to liver, cholesterol catabolism, or cholesterol biosynthesis in liver were compared between SHR-cp and WKY. MATERIALS AND METHODS MaterialsThe Cholesterol E-test Wako was obtained from Wako (Osaka, Japan). The high density lipoprotein (HDL) and very low density lipoprotein (VLDL)/low density lipoprotein (LDL) Quantification Kit was from BioVision, Inc. The Biomasher was obtained from Assist (Tokyo, Japan), QuickGene RNA tissue kit SII from Fujifilm (Tokyo, Japan), and SYBR Ex Script reverse transcription-polymerase chain reaction (RT-PCR) kit from TaKaRa (Shiga, Japan). All other chemicals were of reagent grade and purchased commercially.Animals Male (6-week-old) WKY and SHR-cp were obtained from the Disease Model Co-operative Research Association, Japan. The rats were fed standard chow for 4 weeks and used at 10 weeks of age. The experimental protocol was reviewed and approved by the Animal Care and Use Committee of Fukuyama University.Cholesterol Levels in Liver and Serum One hundred milligrams of liver was homogenized in 500 ml of homogenization buffer (50 mM Tris-HCl, pH 7.5 containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM 2-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and protease inhibitors [1 mM leupeptin, 1 mM pepstatin A, 1 mM chymostatin, and 1 mM antipain]) and centrifuged at 1000ϫg for 10 min. Forty microliters of postnuclear supernatant (PNS) was mixed with 5 ml of Folch extract (chloroformmethanol, 2 : 1), and the mixture was incubated for 10 min at 37°C with shaking. After the mixture was centrifuged at 3000ϫg for 10 min, 3 ml of the supernatant was evaporated dry by boiling at 100°C, and then dissolved in 200 ml of isopropyl alcohol containing 1% Triton-X-100. The cholesterol content of the solution or serum (20 ml) was determined using the Cholesterol E-test Wako (optical density (OD) 600 nm). Isolation of HDL and VLDL/LDL Fractions from SerumThe HDL and VLDL/LD...

Characterization of Fatty Acid Profile in the Liver of SHR/NDmcr-cp (cp/cp) Rats, a Model of the Metabolic Syndrome

“…On the other hand, information on dyslipidemia in SHR/NDcp is very limited. 1,7) To our knowledge, no information is available on fatty acid metabolism in the liver, in particular. To establish SHR/NDcp as a useful model of the metabolic syndrome, it is necessary to document characteristics of fatty acid metabolism in the liver, because hepatic fatty acid composition is considered to be a determinant of insulin sensitivity that acts independently of cellular energy balance and stress.…”

Investigation of dichlorodiphenyltrichloroethane (DDT) on xenobiotic enzyme disruption and metabolomic bile acid biosynthesis in DDT-sprayed areas using wild rats

“…The primer data are summarized in Supplementary Table 2 and some primers used were based on previous reports [2,16,22,23,27,28,31,40,51,52]. Gene expression was normalized using the housekeeping gene (beta-actin) and calculated using the comparative Ct method (ΔΔCT) in both liver and testis.…”