Objective: The endocannabinoid (eCB) system is increasingly recognized as being crucially important in obesity-related hepatic steatosis. By activating the hepatic cannabinoid-1 receptor (CB1R), eCBs modulate lipogenesis and fatty acid oxidation. However, the underlying molecular mechanisms are largely unknown. Methods: We combined unbiased bioinformatics techniques, mouse genetic manipulations, multiple pharmacological, molecular, and cellular biology approaches, and genomic sequencing to systematically decipher the role of the hepatic CB1R in modulating fat utilization in the liver and explored the downstream molecular mechanisms. Results: Using an unbiased normalized phylogenetic profiling analysis, we found that the CB1R evolutionarily coevolves with peroxisome proliferator-activated receptor-alpha (PPARα), a key regulator of hepatic lipid metabolism. In diet-induced obese (DIO) mice, peripheral CB1R blockade (using AM6545) induced the reversal of hepatic steatosis and improved liver injury in WT, but not in PPARα−/− mice. The antisteatotic effect mediated by AM6545 in WT DIO mice was accompanied by increased hepatic expression and activity of PPARα as well as elevated hepatic levels of the PPARα-activating eCB-like molecules oleoylethanolamide and palmitoylethanolamide. Moreover, AM6545 was unable to rescue hepatic steatosis in DIO mice lacking liver sirtuin 1 (SIRT1), an upstream regulator of PPARα. Both of these signaling molecules were modulated by the CB1R as measured in hepatocytes exposed to lipotoxic conditions or treated with CB1R agonists in the absence/presence of AM6545. Furthermore, using microRNA transcriptomic profiling, we found that the CB1R regulated the hepatic expression, acetylation, and transcriptional activity of p53, resulting in the enhanced expression of miR-22, which was found to specifically target SIRT1 and PPARα. Conclusions: We provide strong evidence for a functional role of the p53/miR-22/SIRT1/PPARα signaling pathway in potentially mediating the antisteatotic effect of peripherally restricted CB1R blockade. © 2020 The Authors

Background and Purpose: Obesity, an important risk factor for developing chronic kidney disease (CKD), affects the kidneys by two main molecular signalling pathways: the endocannabinoid/CB1 receptor system, whose activation in obesity promotes renal inflammation, fibrosis, and injury, and the inducible NOS (iNOS), which generates ROS resulting in oxidative stress. Hence, a compound that inhibits both peripheral CB1 receptors and iNOS may serve as an effective therapeutic agent against obesity-induced CKD. Experimental Approach: Here, we describe the effect of a novel peripherally restricted, orally bioavailable dual CB1 receptor/iNOS antagonist, MRI-1867 (3 mg·kg−1), in ameliorating obesity-induced CKD, and compared its metabolic and renal efficacies to a stand-alone peripheral CB1 receptor antagonist (JD5037; 3 mg·kg−1), iNOS antagonist (1400W; 10 mg·kg−1), and pair feeding. Mice with high-fat diet-induced obesity were treated orally with these compounds or vehicle (Veh) for 28 days. Standard diet-fed mice treated with Veh served as controls. Key Results: Enhanced expression of CB1 receptors and iNOS in renal tubules was found in human kidney patients with obesity and other CKDs. The hybrid inhibitor ameliorated obesity-induced kidney morphological and functional changes via decreasing kidney inflammation, fibrosis, oxidative stress, and renal injury. Some of these features were independent of the improved metabolic profile mediated via inhibition of CB1 receptors. An additional interesting finding is that these beneficial effects on the kidney were partially associated with modulating renal adiponectin signalling. Conclusions and Implications: Collectively, our results highlight the therapeutic relevance of blocking CB1 receptors and iNOS in ameliorating obesity-induced CKD. © 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society

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 α-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’s metabolism, thus enhancing its effects by extending its availability to its target cells. © 2019 by the authors.

Background: The endocannabinoid (eCB) system plays a key role in the development of obesity and its comorbidities. Limited information exists on the changes in circulating eCBs following bariatric surgery. Objectives: This study aims to (i) assess the circulating levels of eCBs and related molecules and (ii) examine the association between their levels and numerous clinical/metabolic features pre- and post-operatively. Methods: Sixty-five morbidly obese patients (age 42.78 ± 9.27 years; BMI 42.00 ± 5.01 kg/m 2 ) underwent laparoscopic sleeve gastrectomy (LSG) surgery, and were followed up for 12 months. Data collected included anthropometrics and metabolic parameters. The serum levels of the eCBs, 2-arachidonoylglycerol (2-AG), anandamide (AEA); and their related molecules, arachidonic acid (AA) and oleoylethanolamine (OEA) were measured by liquid chromatography-mass spectrometry. Results: Levels of 2-AG, AEA, and AA were reduced post operatively with no differences in serum OEA levels. The delta changes in eCB levels between pre- and post-operation were correlated with the delta of different metabolic parameters. Positive correlations were found between delta AA and waist circumference (WC) (r = 0.28, P < 0.05), free fat mass (r = 0.26, P < 0.05), SteatoTest score (r = 0.45, P < 0.05), and ALT (r = 0.32, P < 0.05). Delta AEA levels positively correlated with WC (r = 0.30, P < 0.05). Delta 2-AG levels positively correlated with total cholesterol (r = 0.27, P < 0.05), triglycerides (r = 0.55, P < 0.05), and SteatoTest score (r = 0.27, P < 0.05). Delta OEA levels negatively correlated with fasting glucose levels (r = − 0.27, P < 0.05). Conclusions: This study provides compelling evidence that LSG surgery induces reductions in the circulating 2-AG, AEA, and AA levels, and that these changes are associated with clinical benefits related to the surgery including reduced fat mass, hepatic steatosis, glucose, and improved lipid profile. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.

Diabetic nephropathy (DN), a distinct manifestation of diabetic kidney disease, affects approximately 30% of patients with diabetes. While most attention has been focused on glomerular changes related to DN, there is growing evidence that tubulopathy is a key feature in the pathogenesis of this disease. The renal proximal tubule cells (RPTCs) are particularly sensitive to the deleterious effect of chronic hyperglycemia. However, the cellular changes that control the dysfunction of the RPTCs are not fully understood. Controlling glucose reabsorption in the proximal tubules via inhibition of glucose transporters (GLUT) has emerged as a promising therapeutic in ameliorating DN. Overactivation of the renal endocannabinoid (eCB) system via the cannabinoid-1 receptor (CB1R) contributes to the development of DN, and its blockade by globally acting or peripherally restricted CB1R antagonists has been shown to ameliorate renal dysfunction in different murine models for diabetes. Recently, we have utilized various pharmacological and genetic tools to show that the eCB/CB1R system contributes to the development of DN via regulating the expression, translocation, and activity of the facilitative GLUT2 located in the RPTCs. These findings have the potential to be translated into therapy, and support the rationale for the preclinical development of novel renal-specific CB1R and/or GLUT2 inhibitors for the treatment of DN. © 2019 S. Karger AG, Basel. All rights reserved.

Aims: To evaluate the specific role of the endocannabinoid/cannabinoid type-1 (CB1R) system in modulating mitochondrial dynamics in the metabolically active renal proximal tubular cells (RPTCs). Materials and methods: We utilized mitochondrially-targeted GFP in live cells (wild-type and null for the CB1R) and electron microscopy in kidney sections of RPTC-CB1R-/- mice and their littermate controls. In both in vitro and in vivo conditions, we assessed the ability of CB1R agonism or fatty acid flux to modulate mitochondrial architecture and function. Results: Direct stimulation of CB1R resulted in mitochondrial fragmentation in RPTCs. This process was mediated, at least in part, by modulating the phosphorylation levels of the canonical fission protein dynamin-related protein 1 on both S637 and S616 residues. CB1R-induced mitochondrial fission was associated with mitochondrial dysfunction, as documented by reduced oxygen consumption and ATP production, increased reactive oxygen species and cellular lactate levels, as well as a decline in mitochondrial biogenesis. Likewise, we documented that exposure of RPTCs to a fatty acid flux induced CB1R-dependent mitochondrial fission, lipotoxicity and cellular dysfunction. Conclusions: CB1R plays a key role in inducing mitochondrial fragmentation in RPTCs, leading to a decline in the organelle's function and contributing to the renal tubular injury associated with lipotoxicity and other metabolic diseases. © 2018 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.

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. © 2018 American Society for Bone and Mineral Research

Brain trauma was clinically associated with increased osteogenesis in the appendicular skeleton. We showed previously in C57BL/6J mice that mild traumatic brain injury (mTBI) transiently induced bone formation in the femur via the cannabinoid-1 (CB1) receptor. Here, we subjected ICR mice to mTBI and examined the bone response in the skull using microCT. We also measured mast cell degranulation (MCD)72 h post-injury. Finally, we measured brain and calvarial endocannabinoids levels post-mTBI. mTBI led to decreased bone porosity on the contralateral (untouched) side. This effect was apparent both in young and mature mice. Administration of rimonabant (CB1 inverse agonist) completely abrogated the effect of mTBI on calvarial porosity and significantly reduced MCD, compared with vehicle-treated controls. We also found that mTBI resulted in elevated levels of anandamide, but not 2-arachidonoylglycerol, in the contralateral calvarial bone, whereas brain levels remained unchanged. In C57BL/6J CB1 knockout mice, mTBI did not reduce porosity but in general the porosity was significantly lower than in WT controls. Our findings suggest that mTBI induces a strain-specific CB1-dependent bone anabolic response in the skull, probably mediated by anandamide, but seemingly unrelated to inflammation. The endocannabinoid system is therefore a plausible target in management of bone response following head trauma. © 2019, The Author(s).

In this review, we discuss the role of the endocannabinoid (eCB) system in regulating energy and metabolic homeostasis. Endocannabinoids, via activating the cannabinoid type-1 receptor (CB1R), are commonly known as mediators of the thrifty phenotype hypothesis due to their activity in the central nervous system, which in turn regulates food intake and underlies the development of metabolic syndrome. Indeed, these findings led to the clinical testing of globally acting CB1R blockers for obesity and various metabolic complications. However, their therapeutic potential was halted due to centrally mediated adverse effects. Recent observations that highlighted the key role of the peripheral eCB system in metabolic regulation led to the preclinical development of various novel compounds that block CB1R only in peripheral organs with very limited brain penetration and without causing behavioral side effects. These unique molecules, which effectively ameliorate obesity, type II diabetes, fatty liver, insulin resistance, and chronic kidney disease in several animal models, are likely to be further developed in the clinic and may revive the therapeutic potential of blocking CB1R once again. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.