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Over-activation of the endocannabinoid/CB

Obesity is a global medical problem; its common form is known as diet-induced obesity (DIO); however, there are several rare genetic disorders, such as Prader–Willi syndrome (PWS), that are also associated with obesity (genetic-induced obesity, GIO). The currently available therapeutics for treating DIO and GIO are very limited, and they result in only a partial improvement. Cannabidiolic acid (CBDA), a constituent of Cannabis sativa, gradually decarboxylates to cannabidiol (CBD). Whereas the anti-obesity properties of CBD have been reasonably identified, our knowledge of the pharmacology of CBDA is more limited due to its instability. To stabilize CBDA, a new derivative, CBDA-O-methyl ester (HU-580, EPM301), was synthesized. The therapeutic potential of EPM301 in appetite reduction, weight loss, and metabolic improvements in DIO and GIO was tested in vivo. EPM301 (40 mg/kg/d, i.p.) successfully resulted in weight loss, increased ambulation, as well as improved glycemic and lipid profiles in DIO mice. Additionally, EPM301 ameliorated DIO-induced hepatic dysfunction and steatosis. Importantly, EPM301 (20 and 40 mg/kg/d, i.p.) effectively reduced body weight and hyperphagia in a high-fat diet-fed Magel2null mouse model for PWS. In addition, when given to standard-diet-fed Magel2null mice as a preventive treatment, EPM301 completely inhibited weight gain and adiposity. Lastly, EPM301 increased the oxidation of different nutrients in each strain. All together, EPM301 ameliorated obesity and its metabolic abnormalities in both DIO and GIO. These results support the idea to further promote this synthetic CBDA derivative toward clinical evaluation in humans.

Endocannabinoids (eCBs) are internal lipid mediators recognized by the cannabinoid-1 and -2 receptors (CB1R and CB2R, respectively), which also mediate the different physiological effects of marijuana. The endocannabinoid system, consisting of eCBs, their receptors, and the enzymes involved in their biosynthesis and degradation, is present in a vast number of peripheral organs. In this review we describe the role of the eCB/CB1R system in modulating the metabolism in several peripheral organs. We assess how eCBs, via activating the CB1R, contribute to obesity and regulate food intake. In addition, we describe their roles in modulating liver and kidney functions, as well as bone remodeling and mass. Special importance is given to emphasizing the efficacy of the recently developed peripherally restricted CB1R antagonists, which were pre-clinically tested in the management of energy homeostasis, and in ameliorating both obesity- and diabetes-induced metabolic complications.

Oleoyl serine (OS), an endogenous fatty acyl amide (FAA) found in bone, has been shown to have an anti-osteoporotic effect. OS, being an amide, can be hydrolyzed in the body by amidases. Hindering its amide bond by introducing adjacent substituents has been demonstrated as a successful method for prolonging its skeletal activity. Here, we tested the therapeutic efficacy of two methylated OS derivatives, oleoyl &alpha, methyl serine (HU-671) and 2-methyl-oleoyl serine (HU-681), in an ovariectomized mouse model for osteoporosis by utilizing combined micro-computed tomography, histomorphometry, and cell culture analyses. Our findings indicate that daily treatment for 6 weeks with OS or HU-671 completely rescues bone loss, whereas HU-681 has only a partial effect. The increased bone density was primarily due to enhanced trabecular thickness and number. Moreover, the most effective dose of HU-671 was 0.5 mg/kg/day, an order of magnitude lower than with OS. The reversal of bone loss resulted from increased bone formation and decreased bone resorption, as well as reversal of bone marrow adiposity. These results were further confirmed by determining the serum levels of osteocalcin and type 1 collagen C-terminal crosslinks, as well as demonstrating the enhanced antiadipogenic effect of HU-671. Taken together, these data suggest that methylation interferes with OS&rsquo, s metabolism, thus enhancing its effects by extending its availability to its target cells.

Among a multitude of hormonal and metabolic complications, individuals with Prader-Willi syndrome (PWS) exhibit significant bone abnormalities, including decreased BMD, osteoporosis, and subsequent increased fracture risk. Here we show in mice that loss of Magel2, a maternally imprinted gene in the PWS critical region, results in reduced bone mass, density, and strength, corresponding to that observed in humans with PWS, as well as in individuals suffering from Schaaf-Yang syndrome (SYS), a genetic disorder caused by a disruption of the MAGEL2 gene. The low bone mass phenotype in Magel2-/- mice was attributed to reduced bone formation rate, increased osteoclastogenesis and osteoclast activity, and enhanced trans-differentiation of osteoblasts to adipocytes. The absence of Magel2 in humans and mice resulted in reduction in the fatty acid amide bone homeostasis regulator, N-oleoyl serine (OS), whose levels were positively linked with BMD in humans and mice as well as osteoblast activity. Attenuating the skeletal abnormalities in Magel2-/- mice was achieved with chronic administration of a novel synthetic derivative of OS. Taken together, Magel2 plays a key role in modulating bone remodeling and mass in PWS by affecting OS levels and activity. The use of potent synthetic analogs of OS should be further tested clinically as bone therapeutics for treating bone loss. © 2018 American Society for Bone and Mineral Research.

Cannabinoid ligands regulate bone mass, but skeletal effects of cannabis (marijuana and hashish) have not been reported. Bone fractures are highly prevalent, involving prolonged immobilization and discomfort. Here we report that the major non-psychoactive cannabis constituent, cannabidiol (CBD), enhances the biomechanical properties of healing rat mid-femoral fractures. The maximal load and work-to-failure, but not the stiffness, of femurs from rats given a mixture of CBD and Δ(9) -tetrahydrocannabinol (THC) for 8 weeks were markedly increased by CBD. This effect is not shared by THC (the psychoactive component of cannabis), but THC potentiates the CBD stimulated work-to-failure at 6 weeks postfracture followed by attenuation of the CBD effect at 8 weeks. Using micro-computed tomography (μCT), the fracture callus size was transiently reduced by either CBD or THC 4 weeks after fracture but reached control level after 6 and 8 weeks. The callus material density was unaffected by CBD and/or THC. By contrast, CBD stimulated mRNA expression of Plod1 in primary osteoblast cultures, encoding an enzyme that catalyzes lysine hydroxylation, which is in turn involved in collagen crosslinking and stabilization. Using Fourier transform infrared (FTIR) spectroscopy we confirmed the increase in collagen crosslink ratio by CBD, which is likely to contribute to the improved biomechanical properties of the fracture callus. Taken together, these data show that CBD leads to improvement in fracture healing and demonstrate the critical mechanical role of collagen crosslinking enzymes.

Endocannabinoids (eCBs) are internal lipid mediators recognized by the cannabinoid-1 and -2 receptors (CB

The endocannabinoid (eCB) system, including its receptors, ligands, and their metabolizing enzymes, plays an important role in bone physiology. Skeletal cannabinoid type 1 (CB1) receptor signaling transmits retrograde signals that restrain norepinephrine (NE) release, thus transiently stimulating bone formation following an acute challenge, suggesting a feedback circuit between sympathetic nerve terminals and osteoblasts. To assess the effect of chronic in vivo occurrence of this circuit, we characterized the skeletal phenotype of mice with a conditional deletion of the CB1 receptor in adrenergic/noradrenergic cells, including sympathetic nerves. Whereas the deletion of the CB1 receptor did not affect bone mass accrual in the distal femoral metaphysis and in vertebral bodies of young, 12-week-old mice, it substantially increased bone mass in aged, 35-week-old mutant mice as compared to wild-type controls. Contrary to our expectations, specific deficiency of the CB1 receptor in sympathetic neurons led to a markedly increased bone mass phenotype, associated with an enhanced bone formation rate and reduced osteoclastogenesis. Mechanistically, the reduced skeletal eCB 'tone' in the null mice did not reflect in increased sympathetic tone and reduced bone formation, suggesting that constitutive genetic inactivation of sympathetic CB1 receptor disrupts the negative feedback loop between eCBs and NE signaling in bone.

Altered glucose reabsorptionviathe facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CB1R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB1R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB1R blockade or genetically inactivating CB1Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB1R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB1R or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CB1R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN.

Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins. DOI: http://dx.doi.org/10.7554/eLife.05914.001, eLife digest The forces applied to the body during daily activities cause bones to be constantly remodeled, which is essential for keeping them healthy. In most adult organisms, new bone is created at the same rate at which old bone is destroyed. This means that overall bone mass remains the same. But, in diseases such as osteoporosis or bone cancer, bone is destroyed more rapidly than at which new bone is made. This leads to brittle bones that are more likely to fracture. Understanding how to increase the rate of bone renewal might therefore help scientists develop new treatments for bone diseases. Bone is created by cells called osteoblasts and destroyed by other cells called osteoclasts. Both of these types of cells develop from stem cells in the bone marrow. The activity of these cells is controlled by a number of factors, including the matrix of proteins that holds bone together. A group of proteins called galectins are known to act as a bridge between some of the matrix proteins and molecules on the surface of the cells. Vinik et al. took osteoblasts from a mouse skull, grew them in the laboratory, and then exposed them to a galectin protein called galectin-8. This made the osteoblasts release a protein called RANKL, which is known to boost osteoclast activity. When osteoblasts that had been exposed to galectin-8 were grown alongside bone marrow stem cells, more of the stem cells developed into the bone-destroying osteoclasts. Mice that were genetically engineered to produce more galectin-8 than normal mice develop brittle bones, despite also creating new bone at a higher rate than do normal mice. This is because osteoclast activity increases at a greater rate, resulting in an overall loss of bone in these animals. This is similar to what occurs in some individuals with osteoporosis. These experiments therefore suggest that galectin-8 plays an important role in bone remodeling and that it may be a potential target for drugs that treat diseases that weaken bones. DOI: http://dx.doi.org/10.7554/eLife.05914.002

Cannabinoid ligands regulate bone mass, but skeletal effects of cannabis (marijuana and hashish) have not been reported. Bone fractures are highly prevalent, involving prolonged immobilization and discomfort. Here we report that the major non-psychoactive cannabis constituent, cannabidiol (CBD), enhances the biomechanical properties of healing rat mid-femoral fractures. The maximal load and work-to-failure, but not the stiffness, of femurs from rats given a mixture of CBD and Δ(9) -tetrahydrocannabinol (THC) for 8 weeks were markedly increased by CBD. This effect is not shared by THC (the psychoactive component of cannabis), but THC potentiates the CBD stimulated work-to-failure at 6 weeks postfracture followed by attenuation of the CBD effect at 8 weeks. Using micro-computed tomography (μCT), the fracture callus size was transiently reduced by either CBD or THC 4 weeks after fracture but reached control level after 6 and 8 weeks. The callus material density was unaffected by CBD and/or THC. By contrast, CBD stimulated mRNA expression of Plod1 in primary osteoblast cultures, encoding an enzyme that catalyzes lysine hydroxylation, which is in turn involved in collagen crosslinking and stabilization. Using Fourier transform infrared (FTIR) spectroscopy we confirmed the increase in collagen crosslink ratio by CBD, which is likely to contribute to the improved biomechanical properties of the fracture callus. Taken together, these data show that CBD leads to improvement in fracture healing and demonstrate the critical mechanical role of collagen crosslinking enzymes.