They an 02/C02 metabolism-measuring system (model MK-5000). After four

They investigated EPO because of
its nonerythroid effects and because the mechanisms behind these effects remain
unclear. Our study investigated the mechanism underlying EPO’s anti-obesity and
anti-diabetic effects on classical brown adipose tissue (BAT). Researchers used
four-week-old male C57BL/6J mice, who were fed a high-fat diet, and half were
additionally given an intraperitoneal injection of recombinant human EPO (220
IU/kg) thrice a week for four consecutive weeks. A second group of mice were
given a normal chow diet for four weeks. They measured body weight, observed
reduction/production of epididymal and subcutaneous white fat mass, caloric
intake, and locomotor ability.

The HFD-mice were randomly
assigned to groups that were injected intraperitoneally either with 200 IU/kg
of recombinant human EPO (Epoetin Alfa BS injection) or with saline (HFD-Con
group) three times a week for four weeks. Mice designated to the normal chow
diet 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 and
body weight change were monitored once a week. Blood was also drawn once a week
to measure the Hematocrit (Ht) value by centrifuging. At 8 weeks all mice were
euthanized using 50mg/kg injections of sodium pentobarbital. Blood was also
collected by cardiopuncture. Blood plasma was separated via centrifuge for
analysis. They collected and weighed four different tissues; interscapular
brown adipose tissue, subcutaneous white adipose tissue, epididymal white
adipose tissue, and liver tissue.

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All plasma parameters were
measured differently. Blood glucose levels was determined by compact glucose
analyzation. Plasma insulin levels were measured with an ELISA kit. Plasma
triglyceride (TG) as well as total cholesterol levels were measured via Wako
reagents. Hematocrit was measured once a week post treatment delivery. 

Researchers performed a glucose
tolerance test. They did this by EPO or saline control treatments over four
weeks. They then measured blood glucose and insulin from the tail vein. They
did this at 0, 30, 60 and 120 minute marks post glucose injection. Researchers
also measured oxygen consumption. This was analyzed by an 02/C02
metabolism-measuring system (model MK-5000). After four weeks, mice were put in
its chambers where a pump draws in air, and O2 concentration is thereby
measured. Interscapular temperature was also measured, where mice were
anesthetized and skin temperature near iBAT was recorded. Locomotor activity
was measured with a Supermex, post four weeks’ experimentation. Movements of
mice were detected via infrared radiation.

The histological methods
consisted of the four above mentioned tissues fixed in a buffered formalin.
Slides were formed and photomicrographs were taken. This was in order to
measure or evaluate brown adipocytes over randomly chosen areas. MRNA and
microRNA quantifications by Quantitative real-time PCR to analyze MRNA
expression, total RNA was isolated from the four tissue types. Quantitative real-time
PCR was performed with 10 ?M of each primer. Amplification was followed using a
specific protocol. Western blot analysis was done in order to extract proteins
via radioimmunoprecipitation assay lysis buffer. Protein concentrations were
determined with a Protein Quantification Assay kit. Tissue proteins were
resolved on polyacrylamide gels.  All of
the analyses were ran as mean ± SEM. A Student’s t-test was used to compare the
means of two groups. After, repeated ANOVA with post-hoc tests were carried out
in order to produce multiple comparisons. 

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