Characterization of metabolic alterations in the lean metabolically unhealthy alpha defensin transgenic mice.

Inflammation is consistently linked to dysmetabolism. In transgenic mice (Def+/+) model the neutrophilic peptide, alpha defensin, proved atherogenic. This phenotype occurred despite favorable cholesterol and glucose levels, and lower body weight and blood pressure. In this study, integration of metabolic&behavioral phenotyping system, endocrine, biochemical and mitochondrial assessment, pathological and immunohistochemical tests, and multiple challenge tests was established to explore the metabolic impact of alpha defensin. Compared to the control group, Def+/+ mice exhibited lower total energy expenditure and carbohydrate utilization, and higher fat oxidation. Their ACTH-cortisol and thyroid profiles were intact. Intriguingly, they had low levels of glucagon, with high ammonia, uric acid, triglyceride, and lactate. Mitochondrial evaluations were normal. Overall, defensin-induced hypoglucagonemia is associated with lipolysis, restricted glucose oxidation, and enhanced wasting. Def+/+ mice may be a useful model for studying the category of lean, apparently metabolically healthy, and atherosclerotic phenotype, with insight into a potential inflammatory-metabolic link.

Renal Mitochondrial ATP Transporter Ablation Ameliorates Obesity-Induced CKD.

SIGNIFICANCE STATEMENT: This study sheds light on the central role of adenine nucleotide translocase 2 (ANT2) in the pathogenesis of obesity-induced CKD. Our data demonstrate that ANT2 depletion in renal proximal tubule cells (RPTCs) leads to a shift in their primary metabolic program from fatty acid oxidation to aerobic glycolysis, resulting in mitochondrial protection, cellular survival, and preservation of renal function. These findings provide new insights into the underlying mechanisms of obesity-induced CKD and have the potential to be translated toward the development of targeted therapeutic strategies for this debilitating condition. BACKGROUND: The impairment in ATP production and transport in RPTCs has been linked to the pathogenesis of obesity-induced CKD. This condition is characterized by kidney dysfunction, inflammation, lipotoxicity, and fibrosis. In this study, we investigated the role of ANT2, which serves as the primary regulator of cellular ATP content in RPTCs, in the development of obesity-induced CKD. METHODS: We generated RPTC-specific ANT2 knockout ( RPTC-ANT2-/- ) mice, which were then subjected to a 24-week high-fat diet-feeding regimen. We conducted comprehensive assessment of renal morphology, function, and metabolic alterations of these mice. In addition, we used large-scale transcriptomics, proteomics, and metabolomics analyses to gain insights into the role of ANT2 in regulating mitochondrial function, RPTC physiology, and overall renal health. RESULTS: Our findings revealed that obese RPTC-ANT2-/- mice displayed preserved renal morphology and function, along with a notable absence of kidney lipotoxicity and fibrosis. The depletion of Ant2 in RPTCs led to a fundamental rewiring of their primary metabolic program. Specifically, these cells shifted from oxidizing fatty acids as their primary energy source to favoring aerobic glycolysis, a phenomenon mediated by the testis-selective Ant4. CONCLUSIONS: We propose a significant role for RPTC-Ant2 in the development of obesity-induced CKD. The nullification of RPTC-Ant2 triggers a cascade of cellular mechanisms, including mitochondrial protection, enhanced RPTC survival, and ultimately the preservation of kidney function. These findings shed new light on the complex metabolic pathways contributing to CKD development and suggest potential therapeutic targets for this condition.

Hepatic targeting of the centrally active cannabinoid 1 receptor (CB1R) blocker rimonabant via PLGA nanoparticles for treating fatty liver disease and diabetes.

Over-activation of the endocannabinoid/CB1R system is a hallmark feature of obesity and its related comorbidities, most notably type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD). Although the use of drugs that widely block the CB1R was found to be highly effective in treating all metabolic abnormalities associated with obesity, they are no longer considered a valid therapeutic option due to their adverse neuropsychiatric side effects. Here, we describe a novel nanotechnology-based drug delivery system for repurposing the abandoned first-in-class global CB1R antagonist, rimonabant, by encapsulating it in polymeric nanoparticles (NPs) for effective hepatic targeting of CB1Rs, enabling effective treatment of NAFLD and T2D. Rimonabant-encapsulated NPs (Rimo-NPs) were mainly distributed in the liver, spleen, and kidney, and only negligible marginal levels of rimonabant were found in the brain of mice treated by iv/ip administration. In contrast to freely administered rimonabant treatment, no CNS-mediated behavioral activities were detected in animals treated with Rimo-NPs. Chronic treatment of diet-induced obese mice with Rimo-NPs resulted in reduced hepatic steatosis and liver injury as well as enhanced insulin sensitivity, which were associated with enhanced cellular uptake of the formulation into hepatocytes. Collectively, we successfully developed a method of encapsulating the centrally acting CB1R blocker in NPs with desired physicochemical properties. This novel drug delivery system allows hepatic targeting of rimonabant to restore the metabolic advantages of blocking CB1R in peripheral tissues, especially in the liver, without the negative CB1R-mediated neuropsychiatric side effects.

The Metabolic Efficacy of a Cannabidiolic Acid (CBDA) Derivative in Treating Diet- and Genetic-Induced Obesity.

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.

Specific inflammatory osteoclast precursors induced during chronic inflammation give rise to highly active osteoclasts associated with inflammatory bone loss.

Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6ChiCD11blo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α-/- mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.

Renal Proximal Tubule Cell Cannabinoid-1 Receptor Regulates Bone Remodeling and Mass via a Kidney-to-Bone Axis.

The renal proximal tubule cells (RPTCs), well-known for maintaining glucose and mineral homeostasis, play a critical role in the regulation of kidney function and bone remodeling. Deterioration in RPTC function may therefore lead to the development of diabetic kidney disease (DKD) and osteoporosis. Previously, we have shown that the cannabinoid-1 receptor (CB1R) modulates both kidney function as well as bone remodeling and mass via its direct role in RPTCs and bone cells, respectively. Here we employed genetic and pharmacological approaches that target CB1R, and found that its specific nullification in RPTCs preserves bone mass and remodeling both under normo- and hyper-glycemic conditions, and that its chronic blockade prevents the development of diabetes-induced bone loss. These protective effects of negatively targeting CB1R specifically in RPTCs were associated with its ability to modulate erythropoietin (EPO) synthesis, a hormone known to affect bone mass and remodeling. Our findings highlight a novel molecular mechanism by which CB1R in RPTCs remotely regulates skeletal homeostasis via a kidney-to-bone axis that involves EPO.

HU-671, a Novel Oleoyl Serine Derivative, Exhibits Enhanced Efficacy in Reversing Ovariectomy-Induced Osteoporosis and Bone Marrow Adiposity.

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.

Thermoneutrality improves skeletal impairment in adult Prader-Willi syndrome mice.

Human Prader-Willi syndrome (PWS) is characterised by impairments of multiple systems including the growth hormone (GH) axis and skeletal growth. To address our lack of knowledge of the influence of PWS on skeletal integrity in mice, we have characterised the endocrine and skeletal phenotype of the PWS-ICdel mouse model for "full" PWS and determined the impact of thermoneutrality. Tibial length, epiphyseal plate width and marrow adiposity were reduced by 6%, 18% and 79% in male PWS-ICdel mice, with osteoclast density being unaffected. Similar reductions in femoral length accompanied a 32% reduction in mid-diaphyseal cortical diameter. Distal femoral Tb.N was reduced by 62%, with individual trabeculae being less plate-like and the lattice being more fragmented (Tb.Pf increased by 63%). Cortical strength (Ultimate moment) was reduced by 26% as a result of reductions in calcified tissue strength and the geometric contribution. GH and prolactin contents in PWS-ICdel pituitaries were reduced in proportion to their smaller pituitary size, with circulating IGF-1 concentration reduced by 37-47%. Conversely, while pituitary LH content was halved, circulating gonadotropin concentrations were unaffected. Although longitudinal growth, marrow adiposity and femoral geometry were unaffected by thermoneutrality, strengthened calcified tissue reversed weakened cortex of PWS-ICdel femora. While underactivity of the GH-axis may be due to loss of Snord116 expression and impaired limb bone geometry and strength due to loss of Magel2 expression, comprehensive analysis of skeletal integrity in the single gene deletion models is required. Our data imply that thermoneutrality may ameliorate the elevated fracture risk associated with PWS.

Magel2 Modulates Bone Remodeling and Mass in Prader-Willi Syndrome by Affecting Oleoyl Serine Levels and Activity.

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.

The therapeutic potential of targeting the peripheral endocannabinoid/CB1 receptor system.

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.

Age-related regulation of bone formation by the sympathetic cannabinoid CB1 receptor.

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.

Modulation of Renal GLUT2 by the Cannabinoid-1 Receptor: Implications for the Treatment of Diabetic Nephropathy.

Altered glucose reabsorption via the 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.

CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

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.

The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice.

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.

Cannabidiol, a Major Non-Psychotropic Cannabis Constituent Enhances Fracture Healing and Stimulates Lysyl Hydroxylase Activity in Osteoblasts.

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.