antibodies used were specific for atpase subunit  and  ( invitrogen molecular probes ) , monoclonal and polyclonal acetyllysine ( cell signaling technology ) , sirt3 ( as described3 ) , etf and lcad ( generously provided by jerry vockley , university of pittsburgh ) . oxidation of [ 1-c ] palmitic acid by tissue homogenate was adapted from a previously established method26 . briefly , tissue was homogenized in sucrose / tris / edta buffer , and was incubated for 3060 min in the reaction mixture ( ph 8.0 ) , containing [ 1-c ] palmitic acid , and measured for acid - soluble metabolites ( asm ) and trapped co2 . enzymatic activity for lcad was measured using the anaerobic electron transfer flavoprotein ( etf ) fluorescence reduction assay27 using 2 , 6-dimethyheptanoyl - coa as a substrate in recombinant lcad expressed and purified from hek293 t cells with wild - type or catalytically inactive sirt3 , or in e. coli in the absence ( control ) or presence of nicotinamide ( nam , 50 mm)28 .
sirtuins are nad+-dependent protein deacetylases and mediate adaptive responses to a variety of stresses , including calorie restriction and metabolic stress . 
 sirtuin 3 ( sirt3 ) is localized in the mitochondrial matrix where it regulates the acetylation levels of metabolic enzymes , including acetyl coenzyme a synthetase 21,2 . 
 mice lacking both sirt3 alleles appear phenotypically normal under basal conditions , but show marked hyperacetylation of several mitochondrial proteins3 . 
 we report that sirt3 expression is upregulated during fasting in liver and brown adipose tissues . 
 livers from mice lacking sirt3 show higher levels of fatty acid oxidation intermediate products and triglycerides during fasting associated with decreased levels of fatty acid oxidation when compared to wild - type mice . 
 mass spectrometry analysis of mitochondrial proteins shows that long - chain acyl coa dehydrogenase ( lcad ) is hyperacetylated at lysine 42 in the absence of sirt3 . 
 lcad is deacetylated in wild - type mice under fasted conditions and by sirt3 in vitro and in vivo , and hyperacetylation of lcad reduces its enzymatic activity . 
 mice lacking sirt3 exhibit hallmarks of fatty acid oxidation disorders during fasting including reduced atp levels and intolerance to cold exposure . 
 these findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty acid oxidation and demonstrate that sirt3 modulates mitochondrial intermediary metabolism and fatty acid utilization during fasting .