Characterization and regulation of diacylglycerol lipase in isolated nuclei from rat cerebellum

GAVEGLIO, V.L., ; PASQUARÉ, S.J.; GIUSTO, N.M.

Puerto Madryn

Congreso; XLVI Reunión Anual SAIB; 2010

SAIB

Characterization and regulation of diacylglycerol lipase in isolated nuclei from rat cerebellum. Gaveglio, Virginia L., Pasquaré, Susana J. and Giusto, Norma M. Instituto de Investigaciones Bioquímicas de Bahía Blanca, B8000FWB Bahía Blanca, Argentina. E-mail: vgaveglio@criba.edu.ar Using [3H]PtdOH as substrate, we demonstrated the existence of several metabolic pathways which involve phosphatidate phosphohydrolase (LPP), diacylglyceride lipase (DAGL) and monoacylglycerol lipase (MAGL) activities in the nucleus. These activities modulate changes in its lipid composition, generating second messengers implicated in intranuclear signaling. The aim of the present work was to evaluate and characterize DAGL activity using exogenously added [3H] diacylglycerol ([3H]DAG) as substrate. We also studied its regulation by retinoic acid. To this end, adult (4 mo) rat cerebellum was homogenized and highly purified nuclei were isolated by sucrose-density ultracentrifugation. Nuclear preparations were checked for purity by electron microscopy and DAPI stain. Our results indicate that MAG formation increased up to two minutes, 700 μM of substrate and about 50 µg of proteins. Interestingly, MAG production occurred at a higher DAG concentration (150 µM) than that used (60 µM) when DAG was generated from PtdOH. In addition, the nuclear membranes pre-incubated with a nuclear receptor RAR/RXR agonist, all-trans retinoic acid (10 µM), showed a significant DAGL inhibition of about 70%. Taken together, our results demonstrate the presence of an important regulated-enzyme related to signaling pathways in rat cerebellum nuclei.