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Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) cata-lyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCDhas several Ser/ Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD(Ad.MCD) and consti-tutively active AMPK (Ad.CA-AMPK) inH9c2 cells, using an adenoviral gene delivery approach in order to examine if MCDis regulated by AMPK. . | Eur. J. Biochem. 271 2831-2840 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04218.x Malonyl-CoA decarboxylase MCD is differentially regulated in subcellular compartments by 5 AMP-activated protein kinase AMPK Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique Nandakumar Sambandam Michael Steinmetz Angel Chu Judith Y. Altarejos Jason R. B. Dyck and Gary D. Lopaschuk Department of Pediatrics Faculty of Medicine Dentistry University of Alberta Edmonton Canada Malonyl-CoA a potent inhibitor of carnitine pamitoyl transferase-I CPT-I plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase MCD catalyzes the degradation of malonyl-CoA removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD las Severn 1 SSc Thr phosphorylation sites whether it is regulated by AMP-activated protein kinase AMPK has been controversial. We therefore overexpressed MCD Ad-MCDa and conili-tutively active AMPK Ad.CA-AMPK in H9c2 cells using an adenoviral gene delivery approach in order to examine if MCDis regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein Ad.GFP . MCD activity increased 40- to 50-fold in Ad.MCD Ad.GFP cells whnn Compared wíth Ad.GFP control. Co-expressing AMPK with MCD fw-ther augmented MCDexpression and activity in Ad.MCD Ad.CA-AMPK eeHs compared wíth the Ad.MCD Ad.GFP contl ol. subeelhflar fractionation further revealed that 54.7 kDa isofocm of MCD expression was significantly higher in cytosolic fractions of Ad.MCD Ad.CA-AMPK eeUs than of the Ad.MCD Ad.GFP control. However the MCDactivities in cytosolic fractions were not different between the two groups. Interestingly in the mitochondrial fractions MCDactivity significantly increased in Ad.MCD Ad.CA-AMPK cells when compared with Ad.MCD Ad.GFP eeHs. Using phosphoserine and .
