The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese pre-diabetic mice

LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety.España, Ministerio de Sanidad 13/00309 to F.J.B.S. and PI13/00593 to B.R.GConsejería de Salud Junta de Andalucía C-0070-201

Role of Insulin-Growth Factor II on mitochondrial recovery in a cellular model of Parkinson's Disease

Insulin-growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. ROS causes damage to cellular macromolecules affecting several cellular processes and resulting in cell death. Mitochondrial ROS damage has a critical role in the pathobiology of PD. The objective was to assess the IGF-II role in the recovery of the oxidative damage produced on mitochondrial in a cellular model of PD. SN4741 cell line was treated as follows: MPP+ alone, in presence of IGF-II and/or co-incubated BMS (Ins/IGF-I receptors antagonist) or AB (anti-IGF-II-receptor). To assess the effect of IGF-II in the recovery of MPP+ damage, this treatment was removed after 2 h and replaced during another 2 h by medium, IGF-II or IGF-II + BMS or IGF-II + AB. Cell death was analysed through annexin-V Mitochondrial structure, localization and morphology was studied by western blot/ immunochemistry of Mitofilin (Mtf) and electron microscopy; function by Mitotracker and oxygen consumption rate. IGF-II prevented MPP+ cell death. In morphological/structural studies, MPP+ treated cells showed swollen mitochondria with loss of cristae, and electron-lucent matrix, inducing a mitochondrial number reduction. IGF-II retrieved normal-shaped mitochondria with intact cristae and outer/inner membranes. Moreover, MPP+ incubation significantly reduced the expression levels of Mtf compared to the CO. This expression was found in areas that had a very weak mark, indicating mitochondrial destruction or dysfunction. IGF-II coincubation, recovered the expression of Mtf, remaining associated with healthy mitochondrial function. Additionally, the decrease in OCR levels after MPP+ administration was recovered in presence of IGF-II. The BMS-receptor blockage did not modify the IGF-II responses, and AB limited its effect. In conclusion, IGF-II recovers mitochondrial structure and function due to MPP+ damage. This improvement is carried out through the specific IGF-II receptor.Supported by M.G-F.&L.J.S. Proyectos I+D+I-Programa Operativo-FEDER Andalucía 2014-2020 (UMA18-FEDERJA-004) Junta de Andalucía. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Insulin-like growth factor II neuroprotective effects against mitochondrial-oxidative and neuronal damage induced by CORT and MPP+ in dopaminergic neurons

Aims: Parkinson’s disease (PD) affects 1–3% of the population aged over 65. Stress seems to contribute to PD neuropathology, probably by dysregulation of the hypothalamic–pituitary–adrenal axis. Key factors are oxidative stress, mitochondrial dysfunction and neuronal glucocorticoid-induced toxicity. Insulin-like growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Therefore, our aim was to study IGF-II protective effects against oxidative damage on a cellular combined model of PD and mild to moderate stress, based on corticosterone (CORT) and the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Methods: The dopaminergic neuronal cell line SN4741 (RRID:CVCL_S466) derived from mouse substantia nigra were exposed to 200 μM MPP+, 0.5 μM CORT or both, with or without 25 ng/mL IGF-II, for 2.5 or 6 h. Cell viability, oxidative stress parameters, mitochondrial and dopamine markers and intracellular signaling pathways were evaluated. Results: The administration of MPP+ or CORT individually led to cell damage compared to control situations, whereas the combination of both drugs produced very considerable toxic synergistic effect. IGF-II counteracts the mitochondrial-oxidative damage, protecting dopaminergic neurons from death and neurodegeneration. IGF-II promotes PKC activation and nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. Conclusions: IGF-II capacity to protect nigral dopamine neurons against mitochondrial-oxidative damage induced by CORT and MPP+ was demonstrated. Thus, IGF-II is a potential therapeutic tool for prevention and treatment of PD patients suffering mild to moderate emotional stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuroprotective effects of insulin-like growth factor II in a mouse model of Parkinson's disease

Progressive degeneration of the nigrostriatal dopaminergic pathway is a core, currently irreversible pathological hallmark of Parkinson’s disease (PD) that leads to a variety of motor and non-motor symptoms. Here, we aimed to study the potential neuroprotective effects of insulin-like growth factor II (IGF-II) in a PD mouse model based on the chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p), which induces loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNc). Male C57BL6/J mice (N=36) received a 5-week treatment with MPTP/p (or vehicle) and were co-treated with chronic IGF-II (or saline) from either the beginning of the procedure (plus an additional week, days 1-44) or once the MPTP/p insult was already triggered (days 21-44). Baseline and post-treatment measurements for motor performance in the Rotarod and self-grooming in an Open Field were taken. Likewise, dopaminergic (TH, DAT) and neuroinflammatory-related (GFAP) markers in the SNc and the dorsal striatum were studied by immunohistochemistry. Our results revealed that both early and delayed IGF-II co-treatment were successful in preventing motor and behavioral impairment in the MPTP/p model. Moreover, chronic IGF-II protected against MPTP/p-induced loss of dopaminergic neurons in the SNc and promoted a significant recovery of dopaminergic activity in the terminals located in the dorsal striatum, further reducing reactive astrocytosis in these brain regions. Thus, we demonstrated the neuroprotective role of IGF-II in a mouse model of PD, highlighting its potential as a promising therapeutical target for treating this disease. Funding: UMA18-FEDERJA-004, PID2020-113806RB-I00. Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NR5A2/LRH-1 regulates the PTGS2-PGE2-PTGER1 pathway contributing to pancreatic islet survival and function

LRH-1/NR5A2 is implicated in islet morphogenesis postnatally, and its activation using the agonist BL001 protects islets against apoptosis, reverting hyperglycemia in mouse models of Type 1 Diabetes Mellitus. Islet transcriptome profiling revealed that the expression of PTGS2/COX2 is increased by BL001. Herein, we sought to define the role of LRH-1 in postnatal islet morphogenesis and chart the BL001 mode of action conferring beta cell protection. LRH-1 ablation within developing beta cells impeded beta cell proliferation, correlating with mouse growth retardation, weight loss, and hypoglycemia leading to lethality. LRH-1 deletion in adult beta cells abolished the BL001 antidiabetic action, correlating with beta cell destruction and blunted Ptgs2 induction. Islet PTGS2 inactivation led to reduced PGE levels and loss of BL001 protection against cytokines as evidenced by increased cytochrome c release and cleaved-PARP. The PTGER1 antagonist—ONO-8130—negated BL001-mediated islet survival. Our results define the LRH-1/PTGS2/PGE/PTGER1 signaling axis as a key pathway mediating BL001 survival properties.The authors are supported by grants from the Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010 to B.R.G., PI-0085-2013 to P.I.L., PI-0247-2016 to F.J.B.S.), the Consejería de Economía, Innovación y Ciencia (P10.CTS.6359 to B.R.G.), the Ministerio de Ciencia e Innovación co-funded by Fondos FEDER (PI10/00871, PI13/00593 and BFU2017-83588-P to B.R.G and PI17/01004 to F.J.B.S.), Vencer el Cancer (B.R.G), DiabetesCero (B.R.G.) and the Juvenile Diabetes Research Foundation Ltd (17-2013-372 and 2-SRA-2019-837-S-B to B.R.G.). E.M.V. is recipient of a Fellowship from the Ministerio de Ciencia e Innovación co-funded by Fondos FEDER (PRE2018-084907). F.J.B.S. is a recipient of a "Nicolás Monardes" research contracts from Consejería de Salud Junta de Andalucía, (C-0070-2012). A.M.M. is supported by CPII19/00023 and PI18/01590 from the Instituto de Salud Carlos III co-funded by Fondos FEDER. V.C. is supported by a AECC investigator award. CIBERDEM is an initiative of the Instituto de Salud Carlos III

The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese pre-diabetic mice

Romero-Zerbo, Silvana Y. et al.LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety.This work was supported by Consejería de Salud de la Junta de Andalucía, “Andalucía se mueve con Europa” (PI-0574-2012 to S.Y.R.Z.) and Instituto de Salud Carlos III (ISCIII, co-funded by FEDER, EU, “Una manera de hacer Europa”), Ministerio de Sanidad, Gobierno de España (PI13/00309 to F.J.B.S. and PI13/00593 to B.R.G.). S.Y.R.Z. is recipient of a postdoctoral fellowship from Consejeria de Salud de la Junta de Andalucia (RH-0070-2013). I.R.M. is recipient of PFIS predoctoral fellowships from ISCIII (FI11/00636). A.R. received a short-term stay fellowship from Banco Santander (Programa Bolsas Iberoamericanas Jovens Professores e Pesquisadores). FJBS is recipient of a “Miguel Servet II” research contract (CPII13/00042) and also belongs to the regional “Nicolás Monardes” research program of the Consejería de Salud (C-0070-2012; Junta de Andalucía, Spain). CIBERDEM is an initiative of the Instituto de Salud Carlos III. Grupo de trabajo de islotes pancreáticos, Sociedad Española de Diabetes (SED).Peer Reviewe

The synthetic cannabinoids Abn-CBD and LH-21 ameliorate inflammatory status and anxiety in diet-induced diabetic mice

Resumen del póster presentado al XXVIII Congreso Nacional de la Sociedad Española de diabetes, celebrado en Bilbao del 20 al 22 de abril de 2016.[Introduction and objectives]: Novel treatments to fight against type 2 diabetes (T2D) are needed. In this context, recent findings suggest that targeting inflammation is a promising complementary therapy. Indeed, there is evidence suggesting a link between anxiety/depression and obesity/T2D. Abnormal-cannabidiol (Abn-CBD) and LH-21 are synthetic cannabinoids whose pharmacology and effects are still unclear. Abn-CBD seems to activate GPR18 and/or GPR55, whereas LH-21 has been reported to antagonize CB1, though some recent report also suggest GPR55-mediated actions. Interestingly, these GPCRs have been related to inflammatory response and have indeed been found in the CNS. Despite preliminary anti-obesity and anti-hyperglycaemic activity of these drugs, there is a lack of information on the putative modulation of inflammation and anxiety/depression. In order to address these gaps, the effects of chronic administration of Abn-CBD and LH-21 was investigated in diet-induced obese/diabetic mice. [Material and methods]: 10 weeks-old C57Bl/6J mice were fed a high-fat diet (45% kcal content) for 15 weeks. Animals developed an obese/diabetic phenotype with insulin resistance, glucose intolerance, fatty liver, altered inflammatory markers and anxiety. Then, groups of ten mice were daily injected with LH-21 (3 mg/Kg), Abn-CBD (0.05 mg/Kg) or vehicle for two weeks. Food intake and body weight was monitored on a daily basis, glucose handling was assessed by ipGTT and ipITT and finally mice were sacrificed for tissue collection. Several adipokines were determined by ELISA, and inflammatory citokines were assessed by V-PLEX proinflammatory panel kit. Liver histopathology was studied by oil-red staining and F4/80 immunohistochemistry. Behavioural performance was evaluated by the open field test and the elevated plus maze, before and after treatment. [Results]: Abn-CBD and LH-21 treatment did not alter food intake and body weight gain, with little impact on glucose handling. However, both drugs showed anti-inflammatory effects to varying degrees. While Abn-CBD decreased the pro-inflammatory markers IL-6, IL-5 and KC-GRO with no changes in other inflammatory markers, such as IL-2, IL-12p70, IFN-γ, IL-10 and adiponectin, LH-21 decreased plasma levels of leptin without altering any other inflammatory marker. Abn-CBD and LH-21 did not decrease fat content in the liver, nevertheless both drugs decreased macrophages infiltrate in this tissue, suggesting an intra-hepatic anti-inflammatory effect. Regarding behavioural performance, Abn-CBD did not affect locomotion or anxiety. By contrast, LH-21 promoted an anxiolytic behaviour by increasing number of entries into open arms and reverting the obesity/diabetes-induced decrease in time spent and distance travelled in open arms of plus maze, without affecting locomotion. [Conclusions]: Abn-CBD and LH-21 ameliorate the inflammatory status in an animal model of type 2 diabetes, the latter indeed reverting obesity/diabetes-induced anxiety.Funded by the ISCIII (PI13/00309 to FJBS) and the Consejería de Salud de la Junta de Andalucía (PI-0574-2012 to SYRZ).Peer reviewe

The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice

The endocannabinoid system (ECS) is an intercellular signalling mechanism that is present in the islets of Langerhans and plays a role in the modulation of insulin secretion and expansion of the β-cell mass. The downstream signalling pathways mediating these effects are poorly understood. Mammalian target of rapamycin complex 1 (mTORC1) signalling is a key intracellular pathway involved in energy homeostasis and is known to importantly affect the physiology of pancreatic islets. We investigated the possible relationship between cannabinoid type 1 (CB1) receptor signalling and the mTORC1 pathway in the endocrine pancreas of mice by using pharmacological analysis as well as mice genetically lacking the CB1 receptor or the downstream target of mTORC1, the kinase p70S6K1. In vitro static secretion experiments on islets, western blotting, and in vivo glucose and insulin tolerance tests were performed. The CB1 receptor antagonist rimonabant decreased glucose-stimulated insulin secretion (GSIS) at 0.1 µM while increasing phosphorylation of p70S6K1 and ribosomal protein S6 (rpS6) within the islets. Specific pharmacological blockade of mTORC1 by 3 nM rapamycin, as well as genetic deletion of p70S6K1, impaired the CB1-antagonist-mediated decrease in GSIS. In vivo experiments showed that 3 mg/kg body weight rimonabant decreased insulin levels and induced glucose intolerance in lean mice without altering peripheral insulin sensitivity; this effect was prevented by peripheral administration of low doses of rapamycin (0.1 mg/kg body weight), which increased insulin sensitivity. These findings suggest a functional interaction between the ECS and the mTORC1 pathway within the endocrine pancreas and at the whole-organism level, which could have implications for the development of new therapeutic approaches for pancreatic β-cell diseases

The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese pre-diabetic mice

LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety

Insulin-like Growth Factor II Prevents MPP+ and Glucocorticoid Mitochondrial-Oxidative and Neuronal Damage in Dopaminergic Neurons

Stress seems to contribute to Parkinson’s disease (PD) neuropathology, probably by dysregulation of the hypothalamic–pituitary–adrenal axis. Key factors in this pathophysiology are oxidative stress and mitochondrial dysfunction and neuronal glucocorticoid-induced toxicity. The insulin-like growth factor II (IGF-II), a pleiotropic hormone, has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Our aim was to examine the protective effect of IGF-II on a dopaminergic cellular combined model of PD and mild to moderate stress measuring oxidative stress parameters, mitochondrial and neuronal markers, and signalling pathways. IGF-II counteracts the mitochondrial-oxidative damage produced by the toxic synergistic effect of corticosterone and 1-methyl-4-phenylpyridinium, protecting dopaminergic neurons from death and neurodegeneration. IGF-II promotes PKC activation and nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. Thus, IGF-II is a potential therapeutic tool for treatment and prevention of disease progression in PD patients suffering mild to moderate emotional stress