Resat Cinar

Activation of the Nlrp3 inflammasome in infiltrating macrophages by endocannabinoids mediates beta cell loss in type 2 diabetes

Type 2 diabetes mellitus (T2DM) progresses from compensated insulin resistance to beta cell failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. The Nlrp3 inflammasome has been implicated in obesity-induced insulin resistance and beta cell failure. Endocannabinoids contribute to insulin resistance through activation of peripheral CB1 receptors (CB1Rs) and also promote beta cell failure. Here we show that beta cell failure in adult ZDF rats is not associated with CB1R signaling in beta cells, but rather in M1 macrophages infiltrating into pancreatic islets, and that this leads to activation of the Nlrp3-ASC inflammasome in the macrophages. These effects are replicated in vitro by incubating wild-type human or rodent macrophages, but not macrophages from CB1R-deficient (Cnr1−/−) or Nlrp3−/− mice, with the endocannabinoid anandamide. Peripheral CB1R blockade, in vivo depletion of macrophages or macrophage-specific knockdown of CB1R reverses or prevents these changes and restores normoglycemia and glucose-induced insulin secretion. These findings implicate endocannabinoids and inflammasome activation in beta cell failure and identify macrophage-expressed CB1R as a therapeutic target in T2DM.

Convergent translational evidence of a role for anandamide in amygdala-mediated fear extinction, threat processing and stress-reactivity

Endocannabinoids are released ‘on-demand’ on the basis of physiological need, and can be pharmacologically augmented by inhibiting their catabolic degradation. The endocannabinoid anandamide is degraded by the catabolic enzyme fatty acid amide hydrolase (FAAH). Anandamide is implicated in the mediation of fear behaviors, including fear extinction, suggesting that selectively elevating brain anandamide could modulate plastic changes in fear. Here we first tested this hypothesis with preclinical experiments employing a novel, potent and selective FAAH inhibitor, AM3506 (5-(4-hydroxyphenyl)pentanesulfonyl fluoride). Systemic AM3506 administration before extinction decreased fear during a retrieval test in a mouse model of impaired extinction. AM3506 had no effects on fear in the absence of extinction training, or on various non-fear-related measures. Anandamide levels in the basolateral amygdala were increased by extinction training and augmented by systemic AM3506, whereas application of AM3506 to amygdala slices promoted long-term depression of inhibitory transmission, a form of synaptic plasticity linked to extinction. Further supporting the amygdala as effect-locus, the fear-reducing effects of systemic AM3506 were blocked by intra-amygdala infusion of a CB1 receptor antagonist and were fully recapitulated by intra-amygdala infusion of AM3506. On the basis of these preclinical findings, we hypothesized that variation in the human FAAH gene would predict individual differences in amygdala threat-processing and stress-coping traits. Consistent with this, carriers of a low-expressing FAAH variant (385A allele; rs324420) exhibited quicker habituation of amygdala reactivity to threat, and had lower scores on the personality trait of stress-reactivity. Our findings show that augmenting amygdala anandamide enables extinction-driven reductions in fear in mouse and may promote stress-coping in humans.

Cannabinoid 1 Receptor Promotes Cardiac Dysfunction, Oxidative Stress, Inflammation, and Fibrosis in Diabetic Cardiomyopathy

Endocannabinoids and cannabinoid 1 (CB1) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB1 receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH2-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB1, advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT1R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB1 receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB1 receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT1R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB1 receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Endocannabinoids in liver disease

Endocannabinoids are lipid mediators of the same cannabinoid (CB) receptors that mediate the effects of marijuana. The endocannabinoid system (ECS) consists of CB receptors, endocannabinoids, and the enzymes involved in their biosynthesis and degradation, and it is present in both brain and peripheral tissues, including the liver. The hepatic ECS is activated in various liver diseases and contributes to the underlying pathologies. In patients with cirrhosis of various etiologies, the activation of vascular and cardiac CB1 receptors by macrophage‐derived and platelet‐derived endocannabinoids contributes to the vasodilated state and cardiomyopathy, which can be reversed by CB1 blockade. In mouse models of liver fibrosis, the activation of CB1 receptors on hepatic stellate cells is fibrogenic, and CB1 blockade slows the progression of fibrosis. Fatty liver induced by a high‐fat diet or chronic alcohol feeding depends on the activation of peripheral receptors, including hepatic CB1 receptors, which also contribute to insulin resistance and dyslipidemias. Although the documented therapeutic potential of CB1 blockade is limited by neuropsychiatric side effects, these may be mitigated by using novel, peripherally restricted CB1 antagonists. (Hepatology 2011;)

Chronic alcohol produces neuroadaptations to prime dorsal striatal learning

Significance Alcoholism is characterized by a progressive degradation of executive control and an increase in compulsive alcohol seeking that is hypothesized to involve a shift from prefrontal cortex to dorsal striatal (DLS) control over behavior. Here, we show that mice exposed to chronic intermittent alcohol exhibited expansion of dendritic material in DLS neurons, coupled with loss of endocannabinoid CB1 receptor signaling and CB1-mediated long-term depression in the DLS. Behaviorally, chronic alcohol exposure facilitated various forms of DLS-dependent learning and augmented in vivo DLS neuronal activity as correct learned choices were made. These findings support a model in which chronic ethanol causes DLS neuroadaptations that prime for greater striatal control over behavior, potentially contributing to the progression of alcoholism. Drug addictions including alcoholism are characterized by degradation of executive control over behavior and increased compulsive drug seeking. These profound behavioral changes are hypothesized to involve a shift in the regulation of behavior from prefrontal cortex to dorsal striatum (DLS). Studies in rodents have shown that ethanol disrupts cognitive processes mediated by the prefrontal cortex, but the potential effects of chronic ethanol on DLS-mediated cognition and learning are much less well understood. Here, we first examined the effects of chronic EtOH on DLS neuronal morphology, synaptic plasticity, and endocannabinoid-CB1R signaling. We next tested for ethanol-induced changes in striatal-related learning and DLS in vivo single-unit activity during learning. Mice exposed to chronic intermittent ethanol (CIE) vapor exhibited expansion of dendritic material in DLS neurons. Following CIE, DLS endocannabinoid CB1 receptor signaling was down-regulated, and CB1 receptor-dependent long-term depression at DLS synapses was absent. CIE mice showed facilitation of DLS-dependent pairwise visual discrimination and reversal learning, relative to air-exposed controls. CIE mice were also quicker to extinguish a stimulus–reward instrumental response and faster to reduce Pavlovian approach behavior under an omission schedule. In vivo single-unit recording during learning revealed that CIE mice had augmented DLS neuronal activity during correct responses. Collectively, these findings support a model in which chronic ethanol causes neuroadaptations in the DLS that prime for greater DLS control over learning. The shift to striatal dominance over behavior may be a critical step in the progression of alcoholism.

Hepatic cannabinoid‐1 receptors mediate diet‐induced insulin resistance by increasing de novo synthesis of long‐chain ceramides

Obesity is associated with increased activity of two lipid signaling systems (endocannabinoids [ECs] and ceramides), with both being implicated in insulin resistance. Cannabinoid‐1 receptor (CB1R) antagonists reverse obesity and insulin resistance, but have psychiatric side effects. Here we analyzed the role of ceramide in CB1R‐mediated insulin resistance in C57Bl6/J mice with high‐fat diet‐induced obesity (DIO), using JD5037, a peripherally restricted CB1R inverse agonist. Chronic JD5037 treatment of DIO mice reduced body weight and steatosis and improved glucose tolerance and insulin sensitivity. Peripheral CB1R blockade also attenuated the diet‐induced increase in C14:0, C16:0, C18:0, and C20:0 ceramide species with either C16 or C18 sphingosine‐base in the liver. Decreased ceramide levels reflected their reduced de novo synthesis, due to inhibition of the activity of serine‐palmitoyl transferase (SPT) and the expression of its SPTLC3 catalytic subunit, as well as reduced ceramide synthase (CerS) activity related to reduced expression of CerS1 and CerS6. JD5037 treatment also increased ceramide degradation due to increased expression of ceramidases. In primary cultured mouse hepatocytes and HepG2 cells, the EC anandamide increased ceramide synthesis in an eIF2α‐dependent manner, and inhibited insulin‐induced akt phosphorylation by increased serine phosphorylation of IRS1 and increased expression of the serine/threonine phosphatase Phlpp1. These effects were abrogated by JD5037 or the SPT inhibitor myriocin. Chronic treatment of DIO mice with myriocin or JD5037 similarly reversed hepatic insulin resistance, as verified using a euglycemic/hyperinsulinemic clamp. Conclusion: ECs induce CB1R‐mediated, endoplasmic reticulum stress‐dependent synthesis of specific ceramide subspecies in the liver, which plays a key role in obesity‐related hepatic insulin resistance. (Hepatology 2014;58:143–153)

Hyperactivation of anandamide synthesis and regulation of cell-cycle progression via cannabinoid type 1 (CB1) receptors in the regenerating liver

The mammalian liver regenerates upon tissue loss, which induces quiescent hepatocytes to enter the cell cycle and undergo limited replication under the control of multiple hormones, growth factors, and cytokines. Endocannabinoids acting via cannabinoid type 1 receptors (CB1R) promote neural progenitor cell proliferation, and in the liver they promote lipogenesis. These findings suggest the involvement of CB1R in the control of liver regeneration. Here we report that mice lacking CB1R globally or in hepatocytes only and wild-type mice treated with a CB1R antagonist have a delayed proliferative response to two-thirds partial hepatectomy (PHX). In wild-type mice, PHX leads to increased hepatic expression of CB1R and hyperactivation of the biosynthesis of the endocannabinoid anandamide in the liver via an in vivo pathway involving conjugation of arachidonic acid and ethanolamine by fatty-acid amide hydrolase. In wild-type but not CB1R−/− mice, PHX induces robust up-regulation of key cell-cycle proteins involved in mitotic progression, including cyclin-dependent kinase 1 (Cdk1), cyclin B2, and their transcriptional regulator forkhead box protein M1 (FoxM1), as revealed by ultrahigh-throughput RNA sequencing and pathway analysis and confirmed by real-time PCR and Western blot analyses. Treatment of wild-type mice with anandamide induces similar changes mediated via activation of the PI3K/Akt pathway. We conclude that activation of hepatic CB1R by newly synthesized anandamide promotes liver regeneration by controlling the expression of cell-cycle regulators that drive M phase progression.

Inhibitor of Fatty Acid Amide Hydrolase Normalizes Cardiovascular Function in Hypertension without Adverse Metabolic Effects

The enzyme fatty acid amide hydrolase (FAAH) catalyzes the in vivo degradation of the endocannabinoid anandamide, thus controlling its action at receptors. A novel FAAH inhibitor, AM3506, normalizes the elevated blood pressure and cardiac contractility of spontaneously hypertensive rats (SHR) without affecting these parameters in normotensive rats. These effects are due to blockade of FAAH and a corresponding rise in brain anandamide levels, resulting in CB₁ receptor-mediated decrease in sympathetic tone. The supersensitivity of SHR to CB₁ receptor-mediated cardiovascular depression is related to increased G protein coupling of CB₁ receptors. Importantly, AM3506 does not elicit hyperglycemia and insulin resistance seen with other FAAH inhibitors or in FAAH⁻/⁻ mice, which is related to its inability to inhibit FAAH in the liver due to rapid hepatic uptake and metabolism. This unique activity profile offers improved therapeutic value in hypertension.

Overactive cannabinoid 1 receptor in podocytes drives type 2 diabetic nephropathy

Significance Diabetic nephropathy is the leading cause of chronic kidney disease in the United States, and one of the most significant long-term complications of both type 1 and type 2 diabetes, which currently lack fully effective therapy. Hyperglycemia and activation of the renin-angiotensin system (RAS) are thought to be the two main drivers of this pathology. We have recently shown that selective blockade of peripheral cannabinoid receptor-1 (CB1R) delayed and attenuated the development of type 2 diabetes in a rat model. Here we show that the nephropathy-inducing effects of both hyperglycemia and activation of the RAS involve CB1R activation in glomerular podocytes, and that antagonism of peripheral CB1R could represent a novel, effective, and rational approach to both prevent and reverse diabetic nephropathy. Diabetic nephropathy is a major cause of end-stage kidney disease, and overactivity of the endocannabinoid/cannabinoid 1 receptor (CB1R) system contributes to diabetes and its complications. Zucker diabetic fatty (ZDF) rats develop type 2 diabetic nephropathy with albuminuria, reduced glomerular filtration, activation of the renin-angiotensin system (RAS), oxidative/nitrative stress, podocyte loss, and increased CB1R expression in glomeruli. Peripheral CB1R blockade initiated in the prediabetic stage prevented these changes or reversed them when animals with fully developed diabetic nephropathy were treated. Treatment of diabetic ZDF rats with losartan, an angiotensin II receptor-1 (Agtr1) antagonist, attenuated the development of nephropathy and down-regulated renal cortical CB1R expression, without affecting the marked hyperglycemia. In cultured human podocytes, CB1R and desmin gene expression were increased and podocin and nephrin content were decreased by either the CB1R agonist arachydonoyl-2′-chloroethylamide, angiotensin II, or high glucose, and the effects of all three were antagonized by CB1R blockade or siRNA-mediated knockdown of CNR1 (the cannabinoid type 1 receptor gene). We conclude that increased CB1R signaling in podocytes contributes to the development of diabetic nephropathy and represents a common pathway through which both hyperglycemia and increased RAS activity exert their deleterious effects, highlighting the therapeutic potential of peripheral CB1R blockade.

Cannabinoid receptor 1 promotes hepatocellular carcinoma initiation and progression through multiple mechanisms

Hepatocellular carcinoma (HCC) has high mortality and no adequate treatment. Endocannabinoids interact with hepatic cannabinoid 1 receptors (CB1Rs) to promote hepatocyte proliferation in liver regeneration by inducing cell cycle proteins involved in mitotic progression, including Forkhead Box M1. Because this protein is highly expressed in HCC and contributes to its genesis and progression, we analyzed the involvement of the endocannabinoid/CB1R system in murine and human HCC. Postnatal diethylnitrosamine treatment induced HCC within 8 months in wild‐type mice but fewer and smaller tumors in CB1R–/– mice or in wild‐type mice treated with the peripheral CB1R antagonist JD5037, as monitored in vivo by serial magnetic resonance imaging. Genome‐wide transcriptome analysis revealed CB1R‐dependent, tumor‐induced up‐regulation of the hepatic expression of CB1R, its endogenous ligand anandamide, and a number of tumor‐promoting genes, including the GRB2 interactome as well as Forkhead Box M1 and its downstream target, the tryptophan‐catalyzing enzyme indoleamine 2,3‐dioxygenase. Increased indoleamine 2,3‐dioxygenase activity and consequent induction of immunosuppressive T‐regulatory cells in tumor tissue promote immune tolerance. Conclusion: The endocannabinoid/CB1R system is up‐regulated in chemically induced HCC, resulting in the induction of various tumor‐promoting genes, including indoleamine 2,3‐dioxygenase; and attenuation of these changes by blockade or genetic ablation of CB1R suppresses the growth of HCC and highlights the therapeutic potential of peripheral CB1R blockade. (Hepatology 2015;61:1615–1626)