to the various treatment groups n 8 by body weight they are dosed according to body weight at between 7 00 am and 7 45 am with a 1 3 mg kg solution administered intraperitoneally ip orally po or subcutaneously sc the time of dosing is recorded for each group after dosing the rats are returned to their home cages where they then have access to food and water the food consumption is recorded individually every hour for 7 hours and then after 24 h and sometimes 48 h at the end of the experimental session the animals are euthanised the individual data are recorded in microsoft excel sheets outliers are excluded after applying the grubbs statistical evaluation test for outliers and the result is presented graphically using the graphpad prism program assay ii u2014 melanocortin receptor 3 and 5 mc3 and mc5 camp functional assay using the alphascreen u2122 camp detection kit the camp assays for mc3 and mc5 receptors are performed on cells either hek293 or bhk cells stably expressing the mc3 and mc5 receptors respectively the receptors are cloned from cdna by pcr and inserted into the pcdna 3 expression vector stable clones are selected using 1 mg ml g418 cells at approx 80 90 confluence are washed.