They investigated EPO because ofits nonerythroid effects and because the mechanisms behind these effects remainunclear.
Our study investigated the mechanism underlying EPO’s anti-obesity andanti-diabetic effects on classical brown adipose tissue (BAT). Researchers usedfour-week-old male C57BL/6J mice, who were fed a high-fat diet, and half wereadditionally given an intraperitoneal injection of recombinant human EPO (220IU/kg) thrice a week for four consecutive weeks. A second group of mice weregiven a normal chow diet for four weeks. They measured body weight, observedreduction/production of epididymal and subcutaneous white fat mass, caloricintake, and locomotor ability. The HFD-mice were randomlyassigned to groups that were injected intraperitoneally either with 200 IU/kgof recombinant human EPO (Epoetin Alfa BS injection) or with saline (HFD-Congroup) three times a week for four weeks. Mice designated to the normal chowdiet were also randomly assigned to receive either EPO (NC-EPO group) or saline(NC-Con group) injections on the same schedule. The amount of food consumed andbody weight change were monitored once a week.
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Blood was also drawn once a weekto measure the Hematocrit (Ht) value by centrifuging. At 8 weeks all mice wereeuthanized using 50mg/kg injections of sodium pentobarbital. Blood was alsocollected by cardiopuncture. Blood plasma was separated via centrifuge foranalysis.
They collected and weighed four different tissues; interscapularbrown adipose tissue, subcutaneous white adipose tissue, epididymal whiteadipose tissue, and liver tissue.All plasma parameters weremeasured differently. Blood glucose levels was determined by compact glucoseanalyzation.
Plasma insulin levels were measured with an ELISA kit. Plasmatriglyceride (TG) as well as total cholesterol levels were measured via Wakoreagents. Hematocrit was measured once a week post treatment delivery. Researchers performed a glucosetolerance test. They did this by EPO or saline control treatments over fourweeks.
They then measured blood glucose and insulin from the tail vein. Theydid this at 0, 30, 60 and 120 minute marks post glucose injection. Researchersalso measured oxygen consumption. This was analyzed by an 02/C02metabolism-measuring system (model MK-5000). After four weeks, mice were put inits chambers where a pump draws in air, and O2 concentration is therebymeasured.
Interscapular temperature was also measured, where mice wereanesthetized and skin temperature near iBAT was recorded. Locomotor activitywas measured with a Supermex, post four weeks’ experimentation. Movements ofmice were detected via infrared radiation. The histological methodsconsisted of the four above mentioned tissues fixed in a buffered formalin.Slides were formed and photomicrographs were taken. This was in order tomeasure or evaluate brown adipocytes over randomly chosen areas. MRNA andmicroRNA quantifications by Quantitative real-time PCR to analyze MRNAexpression, total RNA was isolated from the four tissue types.
Quantitative real-timePCR was performed with 10 ?M of each primer. Amplification was followed using aspecific protocol. Western blot analysis was done in order to extract proteinsvia radioimmunoprecipitation assay lysis buffer. Protein concentrations weredetermined with a Protein Quantification Assay kit. Tissue proteins wereresolved on polyacrylamide gels.
All ofthe analyses were ran as mean ± SEM. A Student’s t-test was used to compare themeans of two groups. After, repeated ANOVA with post-hoc tests were carried outin order to produce multiple comparisons.