ABSTRACT
Background Recently, several studies have shown that microRNAs are present in high-density lipoprotein, and high-density lipoprotein-microRNA may be a promising disease biomarker. We investigated the stability of high-density lipoprotein-microRNAs in different storage conditions as this is an important issue for its application to the field of clinical research. Methods microRNAs were extracted from the high-density lipoprotein fraction that was purified from the serum. miR-135 a and miR-223, which are known to be present in high-density lipoprotein, were quantified by quantitative real-time PCR. The influence of preanalytical parameters on the analysis of high-density lipoprotein-miRNAs was examined by the effect of RNase, storage conditions, and freezing and thawing. Results The concentrations of microRNA in high-density lipoprotein were not altered by RNase A treatment (0-100 U/mL). No significant change in these microRNAs was observed after storing serum at room temperature or 4â for 0-24 h, and there was a similar result in the cryopreservation for up to two weeks. Also, high-density lipoprotein-microRNAs were stable for, at least, up to five freeze-thaw cycles. Conclusions These results demonstrated that high-density lipoprotein-microRNAs are relatively resistant to various storage conditions. This study provides new and important information on the stability of high-density lipoprotein-microRNAs.
Subject(s)
Cryopreservation , Lipoproteins, HDL/blood , MicroRNAs/blood , Healthy Volunteers , Humans , Lipoproteins, HDL/isolation & purification , Male , MicroRNAs/isolation & purification , Protein Binding , RNA Stability , Real-Time Polymerase Chain Reaction , Refrigeration , Ribonuclease, Pancreatic/chemistryABSTRACT
DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status.
Subject(s)
Carnitine O-Palmitoyltransferase/genetics , DNA Methylation , Fructose/metabolism , Liver/metabolism , PPAR alpha/genetics , Promoter Regions, Genetic , Animals , Lipid Metabolism , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Hippocampal functions such as neuronal protection and synapse formation are positively modulated by neurosteroids, which are synthesized de novo within the brain. However, the mechanisms regulating neurosteroidogenesis remain unclear. Fructose, which is used as a sweetener, affects steroid hormone synthesis in peripheral endocrine organs. This monosaccharide can penetrate the blood-brain barrier and impair hippocampal function. Also, fructose is secreted into milk and is thus delivered to the fetus. Based on these observations, we hypothesized that the hippocampal neurosteroidogenesis in the offspring may be affected by maternal fructose consumption. Female rats were fed with normal water or 20% fructose solution during gestation and lactation. Maternal calorie intake did not change significantly, and no significant change in body weight was observed. The levels of messenger RNAs (mRNAs) for steroidogenic enzymes and proteins in the hippocampus of the offspring were analyzed by real-time reverse transcriptase polymerase chain reaction. Maternal fructose consumption during gestation and lactation increased mRNA levels of P450(11ß)-2, 11ß-HSD-2, and 17ß-HSD-1 in the offspring hippocampus, and reduced levels of mRNAs for StAR, PBR, and 17ß-HSD-3. Maternal fructose consumption might influence hippocampal neurosteroidogenesis in offspring.
