In a recent study published in Molecular Metabolism, researchers investigated whether under chronic [in obese mice with leptin-associated ob gene mutation (ob/ob)] or acute [six hours of fasting in wild-type (wt) mice] reductions in hypothalamic leptin signaling, orexin-A (OX-A)-elicited enhancement in 2-arachidonoyl-glycerol (2-AG) expression led to 2-AG-derived 2-arachidonoyl-sn-glycerol-3-phosphate (2-AGP) production, disassembling alpha-melanocyte-stimulating hormone (α-MSH) anorexigenic inputs, and affecting dietary intake.
Study: Orexin induces the production of an endocannabinoid-derived lysophosphatidic acid eliciting hypothalamic synaptic loss in obesity. Image Credit:khomkrit sangkatechon/Shutterstock.com
Background
Orexin-A (OX-A), a neuropeptide selectively produced by lateral hypothalamic (LH) neurons, controls brain physiology and function by modulating the homeostatic control of energy balance and arousal-associated behaviors.
In the case of acute or chronic brain leptin signaling deficiencies, such as during transient food deprivation and obesity, respectively, Orexin A neuronal activity increases, promoting food-seeking and hyperarousal. However, the leptin-based mechanism is not well-characterized.
Elucidating the adaptive and maladaptive mechanisms that dysregulate the synaptic refinement of orexin-A OX neurons as a response to the balanced or unbalanced energy intake could improve the understanding of behavioral derangements that affect metabolic health during adiposity.
The present study's authors previously demonstrated that orexin-A potently induces the synthesis of 2-AG), an endocannabinoid that increases food intake, promoting hyperphagia and adiposity.
About the study
In the present study, researchers explored OX-A/2-AGP- and leptin-regulated molecular mechanisms, mediating glycogen synthase kinase 3β (GSK-3β)-regulated expression of Tau protein phosphorylation in the threonine 231 epitope (pT231) at the arcuate pro-opiomelanocrotin (POMC)-type neurons of ob/ob and wild-type mice. This was done by modeling mHypoN41 (N41)-type POMC neurons in vitro.
Cell-specific morphological, pharmacological, electrophysiological, and biochemical techniques were combined to elucidate the pathways underlying the loss of POMC-derived α-MSH inputs during impaired or reduced leptin signaling conditions.
The team recruited 60 males (30 non-obese and 30 obese individuals) from Molise University's center for research and training in medicine for aging (CeRMA) in Italy. Individuals with body mass index (BMI) above 30 were considered obese according to the World Health Organization (WHO) criteria. Blood samples were obtained from the participants after overnight fasting to assess serological 2-AGP and OX-A levels.
Male C57BL/6 mice were used for the animal experiments, and polymerase chain reaction (PCR) was performed for genotyping. The murine animals were administered intraperitoneal (IP) infections of vehicle-DPBS, OX-A, leptin, SB334867, PF431396, O-7460, 2-AG-(d8), 2-AGP, and AM095. 5.0 gram-weighing pellets were placed in each cage to assess food consumption, and their weights were assessed the next day.
After the in vivo experiments, brain tissues were obtained for immunohistochemistry (IHC), immunofluorescence, and confocal microscopy analyses. Cell culture experiments were performed using hypothalamic mice mHypoE-N41 neuronal cells.
The density of ppT231-Tau immunoreactivity was assessed. Murine anti-LPA receptor type 1 (LPA1R) antibody specificity was confirmed based on immunoperoxidase reactions. Subsequently, western blot analysis and correlative-light-electron microscopy (CLEM) were performed.
2-AG and 2-AGP levels were determined using liquid chromatography-mass spectrometry (LC-MS), and OX-A levels were determined using enzyme-linked immunosorbent assays (ELISA).
Results
In the case of acute or chronic leptin deficiencies, among short-duration food-deprived mice and obese ob/ob mice, respectively, orexin-A neuronal hyperactivation, enhanced food-seeking, and hyperarousal were observed with the upregulated 2-AG synthesis in the hypothalamus.
Orexin A-elicited increase in 2-AG expression resulted in enhanced production of 2-AG-derived 2-arachidonoyl-sn-glycerol-3-phosphate, a biologically active lysophosphatidic acid (LPA).
2-AGP overproduction occurred in the LH of leptin-deficient murine animals, promoting food consumption by decreasing alpha-melanocyte-stimulating hormone-expressing synaptic input to orexin-A neuronal cells via activating LPA receptor type 1 (LPA1-R).
In addition, 2-AGP upregulation increased pT231-Tau levels in alpha-melanocyte-stimulating hormone projections, regulated by pTyr216-GSK3β signaling pathway activity. 2-AGP and orexin-A expression in obese murine animals and humans strongly correlated.
Leptin deficiency in the murine animals enhanced orexin-A signaling in the arcuate nucleus (ARC), 2-AG overproduction, and elevated 2-AGP levels. In obese humans, upregulation of serological 2-AGP expression was observed. LPA drove feeding by its action at the post-synaptic LPA type 1 receptor in murine POMC neuronal cells.
The lack of leptin and increased OX-A levels increased pT231-Tau levels in POMC neurons through 2-AGP activity. Leptin and 2-AGP affected the phosphorylation of Tau proteins in the ARC by modulating GSK-3β activities in contrasting ways.
2-AGP reduced the excitatory drive onto the lateral hypothalamic orexin-A neurons. Leptin deficiency- and 2-AGP-induced phosphorylation of Tau proteins resulted in the retraction of POMC-derived projections to the lateral hypothalamus.
2-AGP acted as an orexigenic autocrine messenger and induced the retraction of α-MSH inputs to orexin-A neurons through enhanced Tau protein phosphorylation. 2-AG and 2-AGP-regulated remodeling alpha-melanocyte-stimulating hormone projections to orexin-A perikarya that negatively controls synaptic efficacy of melanocortin anorexigenic inputs.
Conclusion
Overall, the study findings highlighted a novel mechanism of regulation of energy homeostasis that explains the decrease in glutamatergic α-MSH inputs to lateral hypothalamic orexin-A neurons in times of leptin deficiency.
Targeting the novel mechanism of hypothalamic synaptic plasticity, which depends on 2-AG metabolism, is independent of cannabinoid receptor type 1 (CB1) expression and might be responsible for hyperphagia after food deprivation and during obesity. This could aid in developing new therapies to manage obesity and associated health disorders.
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