Obesity Affects the Microbiota-Gut-Brain Axis and the Regulation Thereof by Endocannabinoids and Related Mediators.

The hypothalamus regulates energy homeostasis by integrating environmental and internal signals to produce behavioral responses to start or stop eating. Many satiation signals are mediated by microbiota-derived metabolites coming from the gastrointestinal tract and acting also in the brain through a complex bidirectional communication system, the microbiota-gut-brain axis. In recent years, the intestinal microbiota has emerged as a critical regulator of hypothalamic appetite-related neuronal networks. Obesogenic high-fat diets (HFDs) enhance endocannabinoid levels, both in the brain and peripheral tissues. HFDs change the gut microbiota composition by altering the Firmicutes:Bacteroidetes ratio and causing endotoxemia mainly by rising the levels of lipopolysaccharide (LPS), the most potent immunogenic component of Gram-negative bacteria. Endotoxemia induces the collapse of the gut and brain barriers, interleukin 1ß (IL1ß)- and tumor necrosis factor α (TNFα)-mediated neuroinflammatory responses and gliosis, which alter the appetite-regulatory circuits of the brain mediobasal hypothalamic area delimited by the median eminence. This review summarizes the emerging state-of-the-art evidence on the function of the "expanded endocannabinoid (eCB) system" or endocannabinoidome at the crossroads between intestinal microbiota, gut-brain communication and host metabolism; and highlights the critical role of this intersection in the onset of obesity.

Orexin induces the production of an endocannabinoid-derived lysophosphatidic acid eliciting hypothalamic synaptic loss in obesity.

OBJECTIVE: Orexin-A (OX-A) is a neuropeptide produced selectively by neurons of the lateral hypothalamus. It exerts powerful control over brain function and physiology by regulating energy homeostasis and complex behaviors linked to arousal. Under conditions of chronic or acute brain leptin signaling deficiency, such as in obesity or short-term food deprivation, respectively, OX-A neurons become hyperactive and promote hyperarousal and food seeking. However, this leptin-dependent mechanism is still mostly unexplored. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) is known to be implicated in food consumption by promoting hyperphagia and obesity, and we and others demonstrated that OX-A is a strong inducer of 2-AG biosynthesis. Here, we investigated the hypothesis that, under acute (6 h fasting in wt mice) or chronic (in ob/ob mice) hypothalamic leptin signaling reduction, OX-A-induced enhancement of 2-AG levels leads to the production of the 2-AG-derived 2-arachidonoyl-sn-glycerol-3-phosphate (2-AGP), a bioactive lipid belonging to the class of lysophosphatidic acids (LPAs), which then regulates hypothalamic synaptic plasticity by disassembling α-MSH anorexigenic inputs via GSK-3ß-mediated Tau phosphorylation, ultimately affecting food intake. METHODS: We combined cell-type-specific morphological (CLEM and confocal microscopy), biochemical, pharmacological, and electrophysiological techniques to dissect the leptin- and OX-A/2-AGP-mediated molecular pathways regulating GSK-3ß-controlled pT231-Tau production at POMC neurons of obese ob/ob and wild-type (wt) lean littermate mice and in an in vitro model of POMC neurons such as mHypoN41 neurons (N41). RESULTS: 2-AGP is overproduced in the hypothalamus of obese leptin-deficient, or lean 6 h food-deprived mice, and promotes food intake by reducing α-MSH-expressing synaptic inputs to OX-A neurons via lysophosphatidic acid type-1 receptor (LPA1-R) activation, and pT231-Tau accumulation in α-MSH projections. This effect is due to the activation of the Pyk2-mediated pTyr216-GSK3ß pathway and contributes to further elevating OX-A release in obesity. Accordingly, we found a strong correlation between OX-A and 2-AGP levels in the serum of obese mice and of human subjects. CONCLUSIONS: Hypothalamic feeding pathways are endowed with 2-AGP-mediated synaptic plasticity according to their inherent functional activities and the necessity to adapt to changes in the nutritional status. These findings reveal a new molecular pathway involved in energy homeostasis regulation, which could be targeted to treat obesity and related disturbances.

Positive association between plasmatic levels of orexin A and the endocannabinoid-derived 2-arachidonoyl lysophosphatidic acid in Alzheimer's disease.

A regular sleep-wake cycle plays a positive function that preserves synaptic plasticity and brain activity from neuropathological injuries. The hypothalamic neuropeptide orexin-A (OX-A) is central in sleep-wake regulation and has been found to be over-expressed in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) suffering from sleep disturbances. OX-A promotes the biosynthesis of 2-arachidonoylglycerol (2-AG), which, in turn, could be phosphorylated to 2-arachidonoyl lysophosphatidic acid (2-AGP). The reorganization of the actin cytoskeleton during neurite retraction is one of the best-characterized effects of lysophosphatidic acids. However, less information is available regarding the reorganization of the neuronal microtubule network in response to OX-A-induced 2-AG and, possibly consequent, 2-AGP production in AD patients. This is of special relevance also considering that higher 2-AG levels are reported in the CSF of AD patients. Here, we found a positive correlation between OX-A and 2-AGP concentrations in the plasma, and an increase of 2-AGP levels in the CSF of AD patients. Furthermore, a negative correlation between the plasmatic 2-AGP levels and the mini-mental state examination score is also revealed in AD patients. By moving from the human patients to in vitro and in vivo models of AD we investigated the molecular pathway linking OX-A, 2-AG and 2-AGP to the phosphorylation of pT231-Tau, which is a specific early plasma biomarker of this disorder. By LC-MS analysis we show that OX-A, via OX-1R, induces 2-AG biosynthesis via DAGLα, and in turn 2-AG is converted to 2-AGP in primary hippocampal neurons. By confocal microscopy and western blotting assay we found an OX-A- or 2-AGP-mediated phosphorylation of Tau at threonine 231 residue, in a manner prevented by LPA1R (2-AGP receptor) or OX1R (OX-A receptor) antagonism with AM095 or SB334867, respectively. Finally, by patch-clamp recording we documented that 2-AGP-mediated pT231-Tau phosphorylation impairs glutamatergic transmission in the mouse hippocampus. Although further additional research is still required to clarify the potential role of orexin signaling in neurodegeneration, this study provides evidence that counteraction of aberrant OX-A signaling, also via LPA-1R antagonism, may be beneficial in the mild-to-moderate age-related cognitive decline associated with sleep disturbances.

Orexin-A/Hypocretin-1 Controls the VTA-NAc Mesolimbic Pathway via Endocannabinoid-Mediated Disinhibition of Dopaminergic Neurons in Obese Mice.

Disinhibition of orexin-A/hypocretin-1 (OX-A) release occurs to several output areas of the lateral hypothalamus (LH) in the brain of leptin knockout obese ob/ob mice. In this study, we have investigated whether a similar increase of OX-A release occurs to the ventral tegmental area (VTA), an orexinergic LH output area with functional effects on dopaminergic signaling at the mesolimbic circuit. By confocal and correlative light and electron microscopy (CLEM) morphological studies coupled to molecular, biochemical, and pharmacological approaches, we investigated OX-A-mediated dopaminergic signaling at the LH-VTA-nucleus accumbens (NAc) pathway in obese ob/ob mice compared to wild-type (wt) lean littermates. We found an elevation of OX-A trafficking and release to the VTA of ob/ob mice and consequent orexin receptor-1 (OX1R)-mediated over-activation of dopaminergic (DA) neurons via phospholipase C (PLC)/diacylglycerol lipase (DAGL-α)-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). In fact, by retrograde signaling to cannabinoid receptor type 1 (CB1R) at inhibitory inputs to DA neurons, 2-AG inhibited GABA release thus inducing an increase in DA concentration in the VTA and NAc of ob/ob mice. This effect was prevented by the OX1R antagonist SB-334867 (30 mg/Kg, i.p.), or the CB1R antagonist AM251 (10 mg/Kg, i.p.) and mimicked by OX-A injection (40 µg/Kg, i.p.) in wt lean mice. Enhanced DA signaling to the NAc in ob/ob mice, or in OX-A-injected wt mice, was accompanied by ß-arrestin2-mediated desensitization of dopamine D2 receptor (D2R) in a manner prevented by SB-334867 or the D2R antagonist L741 (1.5 mg/Kg, i.p.). These results further support the role of OX-A signaling in the control of neuroadaptive responses, such as compulsive reward-seeking behavior or binge-like consumption of high palatable food, and suggest that aberrant OX-A trafficking to the DA neurons in the VTA of ob/ob mice influences the D2R response at NAc, a main target area of the mesolimbic pathway, via 2-AG/CB1-mediated retrograde signaling.

Orexin-A and endocannabinoids are involved in obesity-associated alteration of hippocampal neurogenesis, plasticity, and episodic memory in mice.

The mammalian brain stores and distinguishes among episodic memories, i.e. memories formed during the personal experience, through a mechanism of pattern separation computed in the hippocampal dentate gyrus. Decision-making for food-related behaviors, such as the choice and intake of food, might be affected in obese subjects by alterations in the retrieval of episodic memories. Adult neurogenesis in the dentate gyrus regulates the pattern separation. Several molecular factors affect adult neurogenesis and exert a critical role in the development and plasticity of newborn neurons. Orexin-A/hypocretin-1 and downstream endocannabinoid 2-arachidonoylglycerol signaling are altered in obese mice. Here, we show that excessive orexin-A/2-arachidonoylglycerol/cannabinoid receptor type-1 signaling leads to the dysfunction of adult hippocampal neurogenesis and the subsequent inhibition of plasticity and impairment of pattern separation. By inhibiting orexin-A action at orexin-1 receptors we rescued both plasticity and pattern separation impairment in obese mice, thus providing a molecular and functional mechanism to explain alterations in episodic memory in obesity.

Orexin-A Prevents Lipopolysaccharide-Induced Neuroinflammation at the Level of the Intestinal Barrier.

In states of intestinal dysbiosis, a perturbation of the normal microbiome composition, the intestinal epithelial barrier (IEB) permeability is increased as a result of the disruption of the epithelial tight junction protein network, in which occludin is mostly affected. The loss of IEB integrity promotes endotoxemia, that is, bacterial lipopolysaccharide (LPS) translocation from the intestinal lumen to the circulatory system. This condition induces an enhancement of pro-inflammatory cytokines, which leads to neuroinflammation through the gut-brain axis. Orexin-A (OX-A), a neuropeptide implicated in many physiological functions and produced mainly in the brain lateral hypothalamic area, is expressed also in several peripheral tissues. Orexin-producing neurons have been found in the myenteric plexus to project to orexin receptor 1 (OX-1R)-expressing enterocytes of the intestinal villi. In the present study we investigated the protective role of OX-A against LPS-induced increase of IEB permeability and microglia activation in both an in vivo and in vitro model of the gut-brain axis. By exploiting biochemical, immunocytochemical, immunohistochemical, and functional approaches, we demonstrate that OX-A preserves the IEB and occludin expression, thus preventing endotoxemia and subsequent neuroinflammation.