Role of the endocannabinoid system in diabetes and diabetic complications

Increasing evidence suggests that an overactive endocannabinoid system (ECS) may contribute to the development of diabetes by promoting energy intake and storage, impairing both glucose and lipid metabolism, by exerting pro‐apoptotic effects in pancreatic beta cells and by facilitating inflammation in pancreatic islets. Furthermore, hyperglycaemia associated with diabetes has also been implicated in triggering perturbations of the ECS amplifying the pathological processes mentioned above, eventually culminating in a vicious circle. Compelling evidence from preclinical studies indicates that the ECS also influences diabetes‐induced oxidative stress, inflammation, fibrosis and subsequent tissue injury in target organs for diabetic complications. In this review, we provide an update on the contribution of the ECS to the pathogenesis of diabetes and diabetic microvascular (retinopathy, nephropathy and neuropathy) and cardiovascular complications. The therapeutic potential of targeting the ECS is also discussed. LINKED ARTICLES: This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issueto

Association of cannabis with glutamatergic levels in patients with early psychosis: Evidence for altered volume striatal glutamate relationships in patients with a history of cannabis use in early psychosis

The associative striatum, an established substrate in psychosis, receives widespread glutamatergic projections. We sought to see if glutamatergic indices are altered between early psychosis patients with and without a history of cannabis use and characterise the relationship to grey matter. 92 participants were scanned: Early Psychosis with a history of cannabis use (EPC\u2009=\u200929); Early Psychosis with minimal cannabis use (EPMC\u2009=\u200925); Controls with a history of cannabis use (HCC\u2009=\u200916) and Controls with minimal use (HCMC\u2009=\u200922). Whole brain T1 weighted MR images and localised proton MR spectra were acquired from head of caudate, anterior cingulate and hippocampus. We examined relationships in regions with known high cannabinoid 1 receptor (CB1R) expression (grey matter, cortex, hippocampus, amygdala) and low expression (white matter, ventricles, brainstem) to caudate Glutamine+Glutamate (Glx). Patients were well matched in symptoms, function and medication. There was no significant group difference in Glx in any region. In EPC grey matter volume explained 31.9% of the variance of caudate Glx (p\u2009=\u20090.003) and amygdala volume explained 36.9% (p\u2009=\u20090.001) of caudate Glx. There was no significant relationship in EPMC. The EPC vs EPMC interaction was significant (p\u2009=\u20090.042). There was no such relationship in control regions. These results are the first to demonstrate association of grey matter volume and striatal glutamate in the EPC group. This may suggest a history of cannabis use leads to a conformational change in distal CB1 rich grey matter regions to influence striatal glutamatergic levels or that such connectivity predisposes to heavy cannabis use

Baked and Buzzed: Investigating the Influence of Co-Use of Cannabis and Alcohol on White Matter Integrity in Emerging Adults

Objective: Growing evidence suggests alcohol and cannabis use independently alter neural structure and functioning, particularly during sensitive developmental time periods such as adolescence and emerging adulthood. However, there has been minimal investigation into the effects co-occurring use of these two substances, despite preliminary evidence of unique acute and psychopharmacological changes due to using alcohol and cannabis together. Method: Data drawn from the IDEAA Consortium was utilized to assess white matter integrity as measured by FreeSurfer’s TRACULA in emerging adults (n=192; 16-27 years old). Timeline Follow-Back was used to calculate past month cannabis use, alcohol use, co-use days, binge alcohol episode, and co-use-binge days. The Stroop task was administered and normed scores were used. Multiple regressions investigated white matter integrity by past month cannabis, alcohol, and co-use days, controlling for appropriate covariates (e.g., site, gender, education, length of abstinence). Analyses were run twice, once with alcohol as measured in standard units and once with binge episodes. Follow-up brain-behavior analyses assessed whether substance use or tracts that differed significantly by substance use then related to Stroop performance. Correction for multiple comparisons was conducted using Benjamini and Hochberg’s (1995) False Discovery Rate correction method. Results: Corrected for multiple comparisons, cannabis use was significantly related to increased mean diffusivity in 12 fronto-limbic and fronto-parietal tracts. Cannabis use also associated with poorer performance on Stroop word reading. Within the MJ+ALC group, increased mean diffusivity associated with better Stroop interference performance. Discussion: The present study found cannabis use was associated with decreased white matter integrity, as measured by mean diffusivity, across fronto-parietal and fronto-limbic tracts. These results suggest a robust relationship between cannabis use and white matter integrity in this neurodevelopmentally sensitive time period. Despite our hypotheses, co-use, alcohol use, and binge drinking did not significantly predict any measures. Future research should further investigate the potential independent and interactive affects of these substances on preclinical and clinical levels. Efforts should be made to inform the public of the likely negative impact of cannabis on white matter quality

Association of cannabis with glutamatergic levels in patients with early psychosis: Evidence for altered volume striatal glutamate relationships in patients with a history of cannabis use in early psychosis

he associative striatum, an established substrate in psychosis, receives widespread glutamatergic projections. We sought to see if glutamatergic indices are altered between early psychosis patients with and without a history of cannabis use and characterise the relationship to grey matter. 92 participants were scanned: Early Psychosis with a history of cannabis use (EPC = 29); Early Psychosis with minimal cannabis use (EPMC = 25); Controls with a history of cannabis use (HCC = 16) and Controls with minimal use (HCMC = 22). Whole brain T1 weighted MR images and localised proton MR spectra were acquired from head of caudate, anterior cingulate and hippocampus. We examined relationships in regions with known high cannabinoid 1 receptor (CB1R) expression (grey matter, cortex, hippocampus, amygdala) and low expression (white matter, ventricles, brainstem) to caudate Glutamine+Glutamate (Glx). Patients were well matched in symptoms, function and medication. There was no significant group difference in Glx in any region. In EPC grey matter volume explained 31.9% of the variance of caudate Glx (p = 0.003) and amygdala volume explained 36.9% (p = 0.001) of caudate Glx. There was no significant relationship in EPMC. The EPC vs EPMC interaction was significant (p = 0.042). There was no such relationship in control regions. These results are the first to demonstrate association of grey matter volume and striatal glutamate in the EPC group. This may suggest a history of cannabis use leads to a conformational change in distal CB1 rich grey matter regions to influence striatal glutamatergic levels or that such connectivity predisposes to heavy cannabis use

Characterizing the Cognitive and Emotional Effects of delta-9-Tetrahydrocannabinol in Distinct Hippocampal Sub-Regions

The objective of this study is to determine the potential differential effects of THC in the DH or VH sub-regions, as well as the upstream effects on PFC neuronal activity and oscillations. Rodents used for electrophysiology were infused with THC or vehicle in the DH or VH regions, combined with PFC recordings. Additionally, a battery of behavioural paradigms was performed. Deficits in short-term memory when THC was infused into both regions was observed, however working memory was impaired with VH infusions only. This could be due to THC-induced dysregulation in the PFC, as beta oscillations were significantly decreased selectively in the VH. Additionally, a selective increase in anxiety-related behaviours was observed following VH THC infusions, but not in the DH, which could be related to changes in ERK 1/2. These findings have implications for how marijuana may differentially impact emotional vs. cognitive functions through differential effects on hippocampal sub-regions

EXPOSURE OF ADOLESCENT MICE TO Δ9-TETRAHYDROCANNABINOL SHAPES IMMUNE RESPONSE IN ADULTHOOD

Marijuana, also called Cannabis, is the illicit drug most frequently used by human adolescents;this is facilitated by the fact that users generally perceive these drugs as relatively harmless. Recent data estimate that cannabis use starts in the period from 12 to 18 years of age, though even earlier use (9\u201310 years) is now anecdotally reported. Among the many consequences related to the cannabis use, it is increasingly emerging as the \u3949-THC, as well as endogenous cannabinoids, are able to affect certain functions of the immune system. In fact, studies conducted in experimental animals indicate that cannabis impairs the immune response by altering the homeostasis of the immune system. The adolescence is a particularly vulnerable period of individual life, both for physical and psychological maturity; also the immune system during this development period might be particularly vulnerable to the effects induced by this drug. However, there aren't currently available studies to investigate whether the cannabis intake during adolescence period can have a negative impact on some immune functions such as the individual's response to infection, allergic and autoimmune pathologies, both during the period of drug intake and in adulthood, thus long time from the last direct cannabis exposure. The purpose of this study was to investigate whether the use of cannabis during adolescence may induce immediate and delayed effects on the immune system that may then persist in adulthood. As immune parameters we considered both innate and acquired immunity, measuring T-cells and macrophage cytokines production. We used Balb C/J male mice, 33 post natal days (PND) as model of adolescence age and 80PND as model of adult age. \u3949-THC was administerd with increasing subcutaneous (s.c.) doses, starting from 5 mg/kg and gradually reaching 15mg/kg in 10 days. Control animals were treated with the vehicle for the same duration of time. Some groups of animals were sacrificed, for the immune evaluation, immediately at the end of the treatment, while other groups of mice were housed until 90 PND before the immune evaluation. We recently added a fourth study group consisting of adult animals treated with the drug, for 10days, and housed for 47 days, up to 137 PND, when the evaluation of immune parameters were performed. The data regarding this last study group are not completed, due to the long times required for the implementation of the experimental protocol and refer only to the innate and acquired immunity. In order to study acquired immunity, young and adult mice were immunized with KLH protein to induce an antigen-specific reaction and Th1/Th2 balance was assessed by measuring the production of IFN-\u3b3, IL-4 and IL-10 by splenic lymphocytes. In order to assess macrophage function we used peritoneal macrophages stimulated in vitro with LPS endotoxin; IL-1\u3b2 and TNF-\u3b1 were assessed as pro-inflammatory cytokines, while IL-10 was evaluated as anti-inflammatory cytokine. The cytokine production was measured by specific ELISA and Real-Time PCR. At the end of \u3949-THC treatment in both young and adult mice, after immunization, a significant reduction in the production of IFN-\u3b3 was observed, while the Th2 cytokines IL-4 and IL-10 were significantly increased. When we measured immune response of adult mice treated with the drug in adolescence, we observed that the production of IFN-\u3b3 was still lower and that, in contrast to what observed immediately at the end of treatment, also the Th2 cytokine levels were decreased compared with vehicle mice. In adult animals, assessed 47 days after the end of treatment, there were no significant differences between \u3949-THC or vehicle groups. We also evaluated the ability of the animals to develop an antibody response by measuring IgM anti-KLH titers in the serum; in all study groups, the antibody titers were significantly reduced compared to the vehicle. Regarding the macrophage function, the \u3949-THC treatment induced in both young and adult mice a significant increase of IL-10 while TNF-\u3b1 and IL-1\u3b2 are decreased. Particularly interesting were the results obtained in mice treated in adolescent age with \u3949-THC respect to vehicle; in fact, we observed an opposite effect since IL-1\u3b2 and TNF-\u3b1 levels were significantly increased while IL-10 production was lower, indicating a switch towards a pro-inflammatory phenotype of the macrophage. These effects on cytokines are at transcriptional level since we observed similar result by measuring IL-10, IL-1\u3b2 and TNF-\u3b1 mRNA with RT-PCR. In adult animals evaluated 47 days after the end of treatment, by analyzing the pro- and anti-inflammatory cytokine levels and their relative mRNA expression, no significant differences were found between treatments considered. In order to understand whether the \u3949-THC effects might be due to an alteration of circulating hormones, we also assessed the modulation of hypothalamic pituitary-adrenal axis (HPA) axis activity, after sub-acute and chronic \u3949-THC treatment, by measuring the corticosterone plasma levels in the three study groups. The corticosterone concentrations were lower in all groups, indicating a long lasting dysregulation of HPA. Using a method HPLC/ Mass Spectrometry, we also evaluated blood levels of \u3949-THC and its main metabolites in the experimental groups considered, showing that at the end of 10 days treatment they were comparable to those that can be found in human heavy smokers. We also demonstrated that in animals treated as adolescents and studied as adults, plasma \u3949-THC was not more present, further assuring that the effects observed were due to a long lasting modulation of immunity In addition, to understand the mechanisms underlying the observed effects, we began to investigate whether the \u3949-THC treatment may affect hematopoietic cells development. Preliminary data obtained by evaluating the number of colonies formed by hematopoietic precursors have not highlighted differences between the different treatment groups. Finally, we started to study also the \u3949-THC effects on neuroinflammation, by evaluating the basal production of pro- and anti-inflammatory cytokines in brain areas that are particularly rich in cannabinoid receptors, such as hippocampus and hypothalamus. Our data indicate that chronic treatment with the drug is able to reduce the basal levels of IL-1\u3b2 and TNF-\u3b1 in the brain areas considered, in adolescent and adult mice assessed immediately at the end of treatment. The basal production of IL-10 instead appears to be increased compared to relative vehicles in mice evaluated immediately at the end of the treatment. In adult animals, treated with the drug as adolescents, the basal production of pro-inflammatory cytokines, in hypothalamus and in hippocampus, appears to be increased with respect to vehicles and IL-10 decreased, indicating a predisposition to a pro-inflammatory status. Although these data are preliminary they indicate a parallel modulation of brain and peripheral cytokines by \u3949-THC. In conclusion, these results indicate that the immune system is profoundly altered by treatments with \u3949-THC, with the presence of a dysregulated response. In particular we can affirm that the administration of \u3949-THC in adolescent animals has significant effects on the immune response that last long after its intake

Behavioral Deficits and Associated Alterations in the Proteome in the Amygdala of Adolescent Rats Exposed to Delta9-Tetrahydrocannabinol as Juveniles

Delta9-tetrahydrocannabinol (THC) is an active component of marijuana. During recent years, the popularity of marijuana in the United States has increased tremendously. Marijuana edibles are a form of marijuana that has become very popular in recent years. These are easily accessible not only to adolescents but also to young children. According to recent statistical data, the consumption of marijuana edibles by children below the age of 5 has increased 600% in the states that have legalized marijuana. This has led to an adverse impact on children’s health as evidenced by a sudden increase in the number of children seeking emergency assistance in hospitals. In the current research, we addressed the issue of possible persistent effects on children’s behavior due to an earlier exposure to THC. Juvenile rats were treated with 10 mg/kg of THC from postnatal day 10 through 16. Once they reached adolescence, these rats were tested using several behavioral paradigms. To evaluate the biological basis for the behavioral deficits observed, brain samples obtained from these rats were subjected to proteomic analysis to determine any altered pathways related to the behavior. Our behavioral data indicated that juvenile exposure to THC has no effect on anxiety-related behavior in adolescents. However, we observed a significant effect of treatment on multiple parameters related to social interactions. Of these, episodes and time of social play were significantly increased in the THC treated rats suggesting alterations in the reward circuit function occurring as a result of developmental THC exposure. In the proteomics, we observed a significant effect on relevant canonical pathways such as the changes in thrombin and opioid signaling. Thrombin signaling in neurons is associated with processes involved in the connection between neurons and opioid signaling is involved in the activation processes of the reward circuit suggesting that juvenile THC exposure alters these processes in adolescence which could have detrimental effects on behavior. Overall, our data suggest that consumption of edibles by juveniles leads to altered behavioral and biochemical outcomes in adolescence. This may be detrimental in terms of the appropriate acquisition of skills necessary for meeting the challenges in future life

Effects of psychological stressors and [delta]�-THC acutely and chronically on zebra finch song behavior and dendritic spine density

This dissertation investigated song performance and dendritic spine density, following acute restraint stress in adult zebra finches and, following chronic mild stress and CB1 receptor partial agonist [delta]�-tetrahydrocannabinol ( [delta]�-THC) treatments (3 mg/kg) during sensorimotor development or adulthood. CB1 receptor agonists and stressors have mechanistic overlap: a stressor activates glucocorticoid corticosterone release in the hypothalamic-pituitary-adrenal axis, and endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are CB1 receptor agonists which operate as an endogenous stress buffer system that turns off the response. The endocannabinoid system is prominent during late postnatal development and may modulate important synaptic fine-tuning. Chronic CB1 receptor agonist treatment or stressors during this developmental stage may disrupt appropriate endocannabinoid signaling mediating brain development. Male zebra finches possess a developmental, sensorimotor critical period for learning a song in a mechanism similar to language acquisition in humans. Initially in sensorimotor development, zebra finches possess a surplus of dendritic spines, which are the anatomical basis of the post-synaptic site with excitatory input and may represent morphological building blocks of learning and memory. Over time, a net elimination occurs as part of the developmental learning process. In this dissertation, acute restraint stress (30 minutes) in adults rapidly increased plasma corticosterone levels, altered performance of spectral and temporal acoustic features, and stimulated dendritic spine and c-Fos immunolabeled nuclei density in higher-order acoustic region NCM. [delta]�-THC, the principal psychoactive component of marijuana, inhibits perceptual sensory processing, and [delta]�-THC pretreatment antagonized the effects of stress on c-Fos density in NCM in a CB1 receptor inverse agonist/antagonist SR141716 (6 mg/kg)-reversible manner. The acute effects differed from chronic effects. In adult groups, chronic mild stress or [delta]�-THC treatments alone did not alter corticosterone levels, song acoustic features, or dendritic spine density in NCM or basal ganglia/striatal region Area X. Both chronic stress and [delta]�-THC treatments during sensorimotor song development resulted in effects persistent into adulthood, with reduced syllable entropy and dendritic spine density in Area X. These effects suggest an interference with typical developmental song learning and brain development. Adolescent brain development may be vulnerable to long-term consequences following chronic exposure to CB1 receptor agonists or stressors, and their effects likely differ than exposure during adulthood. This distinction is important to the elucidation of mechanisms and outcomes of marijuana and psychological disorders, such as depression

The Psychopharmacology of Novel Synthetic Cannabinoids

Over recent years, the rapid proliferation of novel psychoactive substances has presented significant challenges to health professionals, regulators, and forensic scientists alike. Synthetic cannabinoids comprise an increasingly prevalent and diverse class of compounds that are used by many people around the world for recreational purposes. These compounds tend to produce psychoactive effects similar to, but stronger than, those of the prototypical cannabis-derived receptor agonist ∆9-tetrahydrocannabinol. The majority of modern synthetic cannabinoids have never been systematically assessed for their effects in humans, meaning that their psychopharmacological and toxicological effects remain largely uncharacterised. Unfortunately, but perhaps not surprisingly, these compounds are implicated in scores of toxic and fatal episodes worldwide. This thesis presents a series of studies aimed at building new knowledge regarding the behavioural and physiological effects of specific synthetic cannabinoids, their potency and metabolism, their long-term effects on cognitive function and brain neurochemistry, and analytical techniques that may be useful in the development of agonist substitution therapies to assist with synthetic cannabinoid withdrawal. The results obtained in this thesis and the wider literature are combined to identify in vivo structure-activity and structure-metabolism relationships for a wide variety of synthetic cannabinoids. These relationships may prove useful for the prediction of the psychopharmacological properties and metabolic pathways of future novel synthetic cannabinoids, reducing the burden involved in testing large numbers of novel compounds individually. Based on rodent assays, long-lasting cognitive impairments and subtle biochemical modulations are predicted in chronic synthetic cannabinoid users. Finally, analytical techniques for evaluating and monitoring agonist replacement therapy for synthetic cannabinoid withdrawal are established

Molecular Mechanisms of Cannabinoids as Anti-cancer Agents

Cancer is a growing health concern world-wide and is the second most common cause of death after heart diseases. Current treatment strategies such as surgery, chemotherapy and radiation provide some relief to cancer patients but the toxic side effects associated with chemotherapy and radiation often lead to further adverse health effects. Hence there is a need for drugs with better safety profile and improved efficacy. Cannabinoids are a group of compounds with several therapeutic properties and besides their appetite stimulant, anti-emetic and analgesic effects, cannabinoids can inhibit tumor growth, survival and metastasis. The mechanisms of action of cannabinoids as anticancer agents are highly complex and not completely understood. Studies from our laboratory indicate that the specificity protein (Sp) transcription factors, Sp1, Sp3 and Sp4 that belong to the Sp/KLF family of transcription factors are overexpressed in many tumors and regulate critical factors responsible for cancer cell proliferation, growth, angiogenesis and survival. Hence, we hypothesized that cannabinoids elicit their responses on cancer cells by downregulating the expression of Sp proteins and Sp-regulated gene products. Treatment of colon and prostate cancer cells with the cannabinoids WIN and cannabidiol (CBD) inhibited cancer cell proliferation, induced apoptosis and downregulated Sp proteins and Sp-dependent genes. Furthermore, we demonstrated that WIN and CBD-mediated induction of apoptosis and repression of Sp proteins were mediated by phosphatases and that the phosphatase involved in WIN- dependent downregulation of Sp proteins was protein phosphatase 2A (PP2a). In addition WIN induced expression of ZBTB-10, an Sp repressor and downregulated microRNA-27a (miR27a) and these effects were PP2a-dependent indicating that WIN transcriptionally represses Sp protein expression by activating the phosphatase, PP2a. We also investigated the effects of 1,1-bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr) and the 2,2'-dimethyl analog (2,2'-diMeDIM-C-pPhBr), on survivin expression in colon and pancreatic cancer cells. Survivin is an anti-apoptotic protein associated with cancer cell survival and confers radiation-resistance in patients receiving radiotherapy. In addition radiation induces survivin, leading to radioresistance in tumors. In this study we demonstrated that DIM-C-pPhBr and 2,2'-diMeDIM-C-pPhBr inhibit cell proliferation and induce apoptosis in colon and pancreatic cancer cells and in combination with radiotherapy, these drugs suppress radioresistance by inhibiting radiation induced survivin