The Stimulation of Ketogenesis by Cannabinoids in Cultured Astrocytes Defines Carnitine Palmitoyltransferase I as a New Ceramide-Activated Enzyme Cristina Bla ´zquez, Cristina Sa ´nchez, Andre ´s Daza, Ismael Galve-Roperh, and Manuel Guzma ´n Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain Abstract: The effects of cannabinoids on ketogenesis in primary cultures of rat astrocytes were studied.  9 -Tet- rahydrocannabinol (THC), the major active component of marijuana, produced a malonyl-CoA-independent stimu- lation of carnitine palmitoyltransferase I (CPT-I) and ke- togenesis from [ 14 C]palmitate. The THC-induced stimu- lation of ketogenesis was mimicked by the synthetic can- nabinoid HU-210 and was prevented by pertussis toxin and the CB 1 cannabinoid receptor antagonist SR141716. Experiments performed with different cellular modulators indicated that the THC-induced stimulation of ketogene- sis was independent of cyclic AMP, Ca 2+ , protein kinase C, and mitogen-activated protein kinase (MAPK). The possible involvement of ceramide in the activation of ketogenesis by cannabinoids was subsequently studied. THC produced a CB 1 receptor-dependent stimulation of sphingomyelin breakdown that was concomitant to an elevation of intracellular ceramide levels. Addition of ex- ogenous sphingomyelinase to the astrocyte culture me- dium led to a MAPK-independent activation of ketogen- esis that was quantitatively similar and not additive to that exerted by THC. Furthermore, ceramide activated CPT-I in astrocyte mitochondria. Results thus indicate that can- nabinoids stimulate ketogenesis in astrocytes by a mech- anism that may rely on CB 1 receptor activation, sphingo- myelin hydrolysis, and ceramide-mediated activation of CPT-I. Key Words: Astrocytes—Ketone bodies—Carni- tine palmitoyltransferase I—Ceramide—Cannabinoids. J. Neurochem. 72, 1759 –1768 (1999). Cannabinoids, the active components of marijuana, exert a wide spectrum of effects such as alterations in cognition and memory, analgesia, anticonvulsion, anti- inflammation, and alleviation of both intraocular pres- sure and emesis (Abood and Martin, 1992; Pertwee, 1997a). It is currently established that cannabinoids exert their effects by binding to specific plasma membrane receptors (Howlett, 1995; Pertwee, 1997a). To date, two different cannabinoid receptors have been characterized and cloned from mammalian tissues: CB 1 and CB 2 (Howlett, 1995; Pertwee, 1997a). The CB 1 receptor is mainly distributed in the CNS, whereas the CB 2 receptor is mostly expressed in cells of the immune system (Per- twee, 1997a). The recent discovery of a family of en- dogenous ligands of cannabinoid receptors (Hillard and Campbell, 1997; Pertwee, 1997a) and the potential ther- apeutic applications of several cannabinoid ligands (Per- twee, 1997b) have focused much attention on cannabi- noids during the last few years. Astrocytes, the major class of glial cells in the mam- malian brain, play a pivotal role in the regulation of brain energy metabolism by providing neurons with anaple- rotic metabolites and substrates for generation of energy. Studies on metabolic regulation in cultured astrocytes have mostly focused on carbohydrate metabolism (see, e.g. Magistretti and Pellerin, 1996; Wiesinger et al., 1997). However, ketone bodies may replace glucose as the major source of neuronal energy metabolism in pathophysiological situations in which energy depriva- tion ensues (Edmond, 1992; Zammit, 1994). Although the liver is generally believed to be the major organ that supplies extrahepatic tissues with ketone bodies (Zam- mit, 1994), astrocytes in culture are able to produce large amounts of ketone bodies from fatty acids (Auestad et al., 1991; Bla ´zquez et al., 1998) as well as from branched-chain amino acids (Bixel and Hamprecht, 1995). This raises the interesting possibility that astro- cytes may provide neurons with ketone bodies as a glucose-replacing fuel in situ. The existence of a direct and specific action of canna- binoids on glial cells is supported by some recent obser- vations. Thus, astrocytes express the CB 1 receptor mRNA (Bouaboula et al., 1995a) and protein (Sa ´nchez et al., 1998a). Astrocytes in culture have also been shown to bind and take up anandamide, a putative endogenous ligand of the CB 1 receptor (Di Marzo et al., 1994). In Received October 15, 1998; revised manuscript received December 8, 1998; accepted December 8, 1998. Address correspondence and reprint requests to Dr. M. Guzma ´n at Department of Biochemistry and Molecular Biology I, School of Bi- ology, Complutense University, 28040-Madrid, Spain. Abbreviations used: cAMP, cyclic AMP; CPT-I, carnitine palmitoyl- transferase I; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MAPK, mi- togen-activated protein kinase; PMA, 4-phorbol 12-myristate 13- acetate; THC,  9 -tetrahydrocannabinol. 1759 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry