were weaned at age twenty one days and housed individually body weights were obtained at the age of twelve weeks in experiment 2 control offspring derived from wild type parents and knockout offspring derived from knockout parents were weaned at age twenty one days only offspring from litters containing nine to eleven mice were used all others were excluded the males were housed individually and females of similar genotype were housed two per cage body weight perigenital fat pad weights male epididymal fat pads female parametrial fat pads and carcasses were analyzed at the age of fifteen weeks for each individual animal the paired fat pads were combined and weighed total body lipid content was assessed using alcoholic potassium hydroxide digestion with saponification of all fats neutralization and then enzymatic determination of glycerol according to conventional methods salmon and flatt int j obesity 9 443 449 1985 lowell et al nature 366 740 742 1993 male control and knockout animals age ten weeks were housed individually with free access to water and chow the animals were divided and maintained at either room temperature 23 u00b0 c or at 4 u00b0 c three weeks later the interscapular depot of brown fat was obtained and assessed for the following parameters wet weight total protein dna and uncoupling protein ucp content protein and ucp were determined as described lowell et al supra and dna was quantitated using the hoechst dye 33258 and a hoefer fluorometer according to the manufacturer 39 s recommended protocol hoefer scientific instruments adipocytes were isolated from epididymal fat pads of male mice using collagenase.