Endocannabinoid and Orexin-1 Interactions In The Hypothalamus:One Possible Clue To Obesity

Endocannabinoids and CB1 receptors stimulate food deprivation-induced food intake by acting on hypothalamic neurons. Endocannabinoid levels change in the hypothalamus following food deprivation (Kirkham et al., Br. J. Pharmacol., 2002) and in rodents with defective leptin signaling (Di Marzo et al., Nature 2001). Orexin is produced in a distinct subset of neurons of the lateral hypothalamus (LH) projecting to all regions of the brain (Sukurai et al., Celi 1998). Pretreatment with subeffective doses of rimonabant attenuates the orexigenic actions of orexin-1 (Crespo et al., Neuropharm. 2008), whereas electrophysiological data support the inhibitory role of cannabinoids on orexinergic neurons in physiological conditions (Huang et al., J. Neurosci. 2007); although it remains to be clarified if the same phenomenon also occurs in obesity, especially since high neural plasticity occurs in this circuitry for adequate regulation of energy balance (Horvart & Gao, Cell Metab. 2005). We bave investigated here endocannabinoid/orexin-1 interactions in C57B1/6j lean mice and in mice made obese following 14 weeks of a diet-induced obesity (DIO) with 25.5% fat. Immunohistochemical studies (immunoperoxidase, confocal and electron microscopy, single or double-staining) were performed in the LH for orexin-1 (OX-1), CB-1, the endocannabinoid biosynthesising enzymes, DAGL-α and NAPE-PLD, and vescicular GABA or glutamate transporters as markers of GABAergic or glutamatergic fibers. The immunoperoxidase staining was followed by quantitative analysis of each immunoreactive signal using a digital densitometric system (Imaging Computer System, Leica©). Endocannabinoid levels were measured by means of LC-MS. LH orexinergic neurons exhibited DAGL-α immunoreactivity (IR) in the somatodendritic compartment and were immersed in a meshwork of CB 1-positive fibers. Increased DAGL-α , but not NAPE-PLD, was observed both in the LH and arcuate nucleus of DIO vs. lean mice. CB1 IR was localized on both glutamatergic and GABAergic axon terminals surrounding orexinergic neurons. A significant increase of 2-AG, but not of AEA, levels was also found in the hypothalamus of DIO vs. lean mice. The reciprocal cellular and subcellular localization of CB1, GABA, glutamate, DAGL-α and OX-1 in the LH suggests a model of retrograde control by CB1 over OX-1 release. DAGL-α mediated synthesis of 2-AG might be triggered by glutamatergic terminals that innervate OX-1 neurons. In DIO mice, the increase of DAGL-α and 2-AG in orexinergic neurons might cause a postsynaptic disinhibition of orexin release by 2-AG acting on presynaptic CB1-expressing GABAergic neurons and contribute to hyperphagia. Electron microscopy observations and electrophysiological experiments are ongoing to substantiate this hypothesis.