Endocannabinoids in Islets of Langerhans: The Ugly ...

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Endocannabinoids in Islets of Langerhans: The Ugly, the Bad and the Good Facts

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Isabel González-Mariscal, Josephine M. Egan

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Laboratory of Clinical Investigation, National Institute on Aging (NIA), National Institutes

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of Health (NIH), Baltimore, MD 21224, United States of America.

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Correspondence should be addressed to:

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Isabel González-Mariscal, Ph.D.

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Laboratory of Clinical Investigation, National Institute on Aging (NIA), National Institutes

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of Health (NIH), Baltimore, MD 21224, United States of America.

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Tel: (410) 558-8414; Fax: (410) 558-8381

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Email: [email protected]

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Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (066.078.062.146) on May 3, 2018. Copyright © 2018 American Physiological Society. All rights reserved.

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The endocannabinoid system (ECS) is an evolutionary ancient signaling network

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involved

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endocannabinoids (ECs), anandamine (AEA) and 2-arachidonoyl glycerol (2-AG), their

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G protein-coupled receptors, cannabinoid 1 (CB1R) and 2 (CB2R), the non-classical

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GPR55, and the enzymes responsible for synthesis and degradation of ECs (34, 35)

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(Figure 1).

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ECs are synthesized on demand by the enzymes N-acyl phosphatidylethanolamine

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phospholipase D (NAPE-PLD; AEA synthesis) and diacylglycerol lipase (DAGL; 2-AG

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synthesis) from membrane bound arachidonate-based precursors (34). Once secreted

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and internalized the ECs are rapidly degraded by fatty acid amide hydrolase (FAAH)

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and monoacylglycerol lipase (MAGL) and the breakdown products are recycled (34)

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(Figure 1A). In obesity, as happens in westernized diets that are rich in fats and sugars,

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the ECS becomes overactive by primarily increasing the synthesis of ECs (8, 39).

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The CBRs are present in the plasma membrane of many organs (Figure 1B) regulating

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a plethora of functions. While CB2R is primarily in immune cells (4, 18), CB1R is

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abundant in brain, where it controls pre-synaptic retrograde inhibition of excitation, and

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regulates appetite and the reward response in hypothalamus. In the periphery, it

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regulates gut motility (52) and incretin (GIP and GLP-1) secretion in the intestines.

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CB1R is expressed in other tissues with endocrine functions, such as adrenal glands,

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ovaries, testicles, and vas deferens (10). In the endocrine pancreas, CB1R activation by

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autonomous EC synthesis and autocrine action in beta-(β-)cells serves as a negative

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feedback loop to many β-cell functions (19, 26).

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The CBRs are Gαi/o protein-coupled and inhibit adenylyl cyclase (AC) activation and

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cAMP-protein kinase A (PKA) activity. They also activate mitogen-activated protein

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kinases (MAPK) and inhibit voltage-gated L- N- and P/Q-type Ca2+ channels and

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inwardly rectifying K+ channels, leading to inhibition of signal transmission and

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diminished release of secretory products from β-cells (19,20). In liver and in β-cells,

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CB1R activation negatively modulates the insulin receptor (IR) pathway by a direct

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interaction of its Gαi subunit with the β subunit of IR (27), and diminished ligand-

in

maintaining

cellular

homeostasis

(15).

It

is

composed

of

two


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mediated CB1R activation improves insulin action (12, 33). We will discuss the literature

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as it relates to modulation of islet function.

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The ECS in islets.

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The Conflicts - the Ugly

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Conflict #1. Which islet cells contain CBRs and which synthesize ECs? Lack of antibody

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specificity has resulted in conflicting reports as to which islet cells express CBRs.

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Evidence favors that the ECS is not expressed in either acinar or ductal tissue of mature

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pancreas. In the endocrine pancreas, CB1R has been variously reported to be present

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in α-, δ- and β-cells (7, 24, 26, 43, 56). Recently we found by molecular analysis of

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single cells disaggregated from human islets that CB1R is expressed in all β-cells but

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not α- or δ-cells (19). We and others also reported that isolated islets contain an

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autonomous ECS (7, 26): ECs are synthesized on demand in response to glucose in a

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concentration-dependent manner, with 2-AG being the most abundant EC (7, 26, 39).

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During development, ECs influence the final adult architecture of islets (38), indicating a

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developmental role of the ECS in islets.

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Conflict #2. Are there differences between rodent and human expression of CBRs in

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islets that add to uncertainty? The human CB1R gene translates into 3 different protein

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isoforms that have varying ligand affinity and tissue-specific distribution (19, 49, 53, 55).

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Human β-cells and hepatocytes abundantly express CB1b, a shorter isoform that is

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virtually absent in brain (19). The existence of isoforms that could be preferably targeted

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introduces a novel variable into the therapeutic equation. Pharmacological approach to

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targeting CB1R activity in the periphery have been problematic because of the lipophilic

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nature of CBR modifiers that freely cross the blood brain barrier.

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Conflict #3. What is the evidence for the presence of other CBRs besides CB1R in

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islets? Whole islets contain resident macrophages, blood vessels and nerve terminals,

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and express transcripts for CB2R but to a much lower extent (100-fold difference) than

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CB1R (7, 16, 24). The CB2R synthetic antagonist AM630, in pharmacological amounts,

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lessened the EC-induced reduction of intracellular Ca+2 concentration oscillations in

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islets in a pertussis toxin-dependent manner (24). The CB2R synthetic agonist JWH133

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reduced insulin secretion from isolated mouse and human islets (7, 24), while JWH015,


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another synthetic CB2R agonist, was reported to increase glucose-stimulated insulin

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secretion from isolated rat islets (57). In mice, JWH133 reduced the glycemic levels in

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blood after a glucose load, while AM630 had the opposite effect (6). There is therefore

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conflict especially between in vivo and in vitro results with the use of CB2R modifying

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agents. However, a CB2R agonist did not prevent macrophage infiltration in islets of

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high fat/streptozotocin (STZ)-induced diabetic mice (57) as has been shown to occur

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with CB1R blockade (22, 23).

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GPR55 is highly expressed in brain (50) and signals through Gα13 and Gq proteins (29).

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Many endogenous and synthetic compounds have been found to bind it, including

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cannabinoids such as THC and AEA in nanomolar concentrations (2, 48). Its

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endogenous ligands appear to be lysophosphatidylinositol and its 2-arachidonoyl

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derivative, 2-arachidonoyl lysophosphatidylinositol. In the periphery it has been reported

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to be expressed in white adipose tissue, liver, gastrointestinal tract and islets (21, 46)

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(Figure 1B). Activation of GPR55 by O-1606, a synthetic agonist, increased glucose-

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stimulated Ca2+ concentration mobilization in β-cells and insulin secretion, and, based

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on immunohistochemistry, GPR55 co-localizes with insulin staining (46). Other agonists

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of GPR55, such as Abn-CBD, were reported to enhance insulin secretion from an

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insulinoma cell line (40). In vivo in rodents, activation of GPR55 leads to improved

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glucose tolerance and increased glucose-stimulated plasma insulin levels (40, 46).

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GPR55 not only is involved in β-cell function, but comparable to CB1R, it is also

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involved in maintaining β-cell mass. Blockade of GPR55 reduced cell proliferation and

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survival, and caused a metabolic shift towards oxidative phosphorylation, reducing

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lactate and carnitine production and PI3K-Akt signaling (9). In STZ-induced diabetic

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mice, treatment with Abn-CBD increased β-cell numbers in islets and reduced

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circulating glucose levels, while it increased plasma insulin levels; these effects were

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dependent on the presence of GLP-1R and GIPR since mice with generic knockout of

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those receptors had no change in glucose or insulin levels when treated with Abn-CBD,

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compared to vehicle (41). Any influence of physiological levels of endogenous ligands

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on GPR55 activity in β-cells has not been forthcoming.

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Of note, heterodimers of the cannabinoid receptors have been described that impact on

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their intracellular signaling (3, 11, 25). The existence of these interactions, although not


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described yet in β-cells, may complicate the study of individual receptors: future studies

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of such possible interactions are warranted.

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Conflict #4. Are ECs promiscuous, i.e., is there one ligand for one receptor? AEA has

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higher affinity for CB1R than for CB2R while 2-AG binds equally to both CB1R and

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CB2R. But the ECs also seem to activate other receptors, including GPR55, in the

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nanomolar range (45). Accepting that all three receptors are present within islets, using

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exogenous ECs to study the specific role of CB1R may not be the best approach. More

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specific alternatives exist when using synthetic ligands such as ACEA, although there

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are some such as AM251, SR141716A or the novel compound LH-21, which are known

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CB1R antagonists that also have agonism activity for GPR55 (14, 42, 45, 47).

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Furthermore, differences in the binding affinity between species exist (55) and need to

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be taken into consideration when studying their effect on human vs. rodent islets.

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Additionally, recent data show that other receptors such as the transient receptor

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potential vanilloid 1 (TRPV1) that are activated by cannabinoids (58), are involved in

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regulating intracellular Ca+2 levels and therefore impact islet function (1, 36).

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Importantly, pancreatic β‐ and α‐cells express key enzymes for AEA metabolism (37,

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54) and using cell cultures of mixed populations or using whole islets cannot give the full

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picture of the role of CB1Rs in just β-cells, or indeed of any individual receptor.

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Moreover, substantial levels of circulating AEA are found in blood (39), which can

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confound findings of islet endogenous ECs.

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Not only ECs signal through various receptors, but also signal through different subunits

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of the CBRs. Besides signaling through Gαi subunit, a few ligands, in micromolar

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ranges, signal through the Gq subunit of CB1R in hippocampal neurons, which couples

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to phospholipase C to release intracellular Ca2+ and causes depolarization (28). In fact,

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in islets of Langerhans, micromolar ranges of synthetic and endogenous CB1R agonists

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induce insulin secretion (30–32), indicating that supraphysiologic concentrations of

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cannabinoids may also signal through the Gq subunit in β-cells.

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β-Cell Dysfunction - the Bad

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Early studies led us to conclude that activated CB1Rs are simply negative regulators of

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AC, thereby diminishing stimuli to insulin secretion, such as incretins and pituitary AC-


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activating peptide that are reliant of AC activation (Figure 2). However, a more complex

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picture is emerging, implicating their influence on K+ and Ca2+ ion channels, MAPK

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signaling, ceramide synthesis, mitochondrial function and Akt signaling (20, 27).

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Additionally, CB1R in β-cells has an impact on cell viability. By direct interaction of

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activated CB1R with IRs, Akt and Bad phosphorylation are reduced (27), leading to

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reduced proliferation and increased β-cell death. In opposition, ablation of CB1R in β-

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cells prevents diet-induced intra-islet oxidative stress production and reduces the

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activation of the MAPK pathway, thereby reducing high glucose and palmitate-induced

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Nlrp3 inflammosome activation and caspase 3 cleavage, resulting in preservation of

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islet viability (20) (Figure 2). Furthermore CB1R is involved in an indirect manner in

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islets viability: CB1R on resident islet and pancreatic macrophages, when activated,

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upregulates the Nlrp3 inflammasome and enhances IL-1β secretion from macrophages,

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thereby it is involved in diet-induced islet inflammation and β-cell dysfunction (22, 23). In

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westernized diets, constant β-cell stimulation due to persistent dysglycemia occurs.

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Unremitting stimuli not only increase insulin secretion but also increase local EC levels.

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Furthermore, elevated levels of ECs, by stimulating CB1R in macrophages and in β-

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cells, activate the inflammatory response, causing β-cell dysfunction and apoptosis.

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The good

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We have discussed that β cells contain an autonomous ECS, and ECs are synthesized

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on demand in response to glucose stimulation and act in an autocrine fashion. This form

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of signaling works as a negative feedback to avoid hypoglycemia and maintain

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homeostasis in insulin secretion. Furthermore, it seems that the ECS in β-cells have

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evolved to protect against inflammation that would potentially be deleterious to β-cells.

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CB1R in β-cells functions as a double-edged sword: 1) when acutely activated it guards

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against over-activity of β-cells, 2) but in obese conditions, the ECs in circulation and in

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islets are chronically increased and their activation of CB1R even when β-cells are not

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in a post-prandial state eventually impedes β-cell function, leading to islet inflammation

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and enhanced β-cell apoptosis. CB1R antagonism reduces diet-induced inflammation in

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islets (22, 23, 47), enhances phosphorylation of Akt and Bad, and increases mTORC1

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signaling (5, 27), thereby favoring β-cell turnover and cell viability, and preventing

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apoptosis, and could be used as a therapy for the treatment of diabetes. However,


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activation of CBRs is a potent therapy for some forms of cancer, depending on receptor

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expression, since activation of CB1 may reduce cell proliferation and induce apoptosis

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(44). Individual-based and tissue-specific therapies will need to be taken into account

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when considering the ECS as a therapeutic target.

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Conclusions

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The ECS is a potential target for novel therapies that protect islets from inflammation

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and apoptosis. In the early 2000s rimonabant became available for treating obesity (13,

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17, 51), and although promising metabolic outcomes were achieved, adverse

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psychiatric effects occurred. Perhaps central nervous system effects can be overcome

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by a second generation of synthetic CB1R modifiers that do not cross the blood brain

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barrier. Furthermore, isoforms of CB1R could be novel targets for drug design, including

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Morpholino oligos to target specific splice variants. Alterations in types of dietary fats

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can also be considered so as to decrease EC synthesis from precursors.


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REFERENCES 1.

Akiba Y, Kato S, Katsube K, Nakamura M, Takeuchi K, Ishii H, Hibi T. Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet β cells modulates insulin secretion in rats. Biochem Biophys Res Commun 321: 219–225, 2004.

184 185

2.

Baker D, Pryce G, Davies WL, Hiley CR. In silico patent searching reveals a new cannabinoid receptor. Trends Pharmacol Sci 27: 1–4, 2006.

186 187 188 189 190

3.

Balenga NA, Martínez-Pinilla E, Kargl J, Schröder R, Peinhaupt M, Platzer W, Bálint Z, Zamarbide M, Dopeso-Reyes IG, Ricobaraza A, Pérez-Ortiz JM, Kostenis E, Waldhoer M, Heinemann A, Franco R. Heteromerization of GPR55 and cannabinoid CB 2 receptors modulates signalling. Br J Pharmacol 171: 5387– 5406, 2014.

191 192

4.

Basu S, Dittel BN. Unraveling the complexities of cannabinoid receptor 2 (CB2) immune regulation in health and disease. Immunol Res 51: 26–38, 2011.

193 194 195 196

5.

Bermudez-Silva FJ, Romero-Zerbo SY, Haissaguerre M, Ruz-Maldonado I, Lhamyani S, El Bekay R, Tabarin A, Marsicano G, Cota D. The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice. Dis Model Mech 9: 51–61, 2016.

197 198 199

6.

Bermudez-Silva FJ, Sanchez-Vera I, Suárez J, Serrano A, Fuentes E, JuanPico P, Nadal A, Rodríguez de Fonseca F. Role of cannabinoid CB2 receptors in glucose homeostasis in rats. Eur J Pharmacol 565: 207–211, 2007.

200 201 202 203

7.

Bermúdez-Silva FJ, Suárez J, Baixeras E, Cobo N, Bautista D, Cuesta-Muñoz AL, Fuentes E, Juan-Pico P, Castro MJ, Milman G, Mechoulam R, Nadal A, Rodríguez de Fonseca F. Presence of functional cannabinoid receptors in human endocrine pancreas. Diabetologia 51: 476–87, 2008.

204 205 206

8.

Bermudez-Silva FJ, Viveros MP, McPartland JM, Rodriguez de Fonseca F. The endocannabinoid system, eating behavior and energy homeostasis: The end or a new beginning? Pharmacol Biochem Behav 95: 375–382, 2010.

207 208 209 210

9.

Bernier M, Catazaro J, Singh NS, Wnorowski A, Boguszewska-Czubara A, Jozwiak K, Powers R, Wainer IW. GPR55 receptor antagonist decreases glycolytic activity in PANC-1 pancreatic cancer cell line and tumor xenografts. Int J Cancer 141: 2131–2142, 2017.

211 212 213

10.

Cacciola G, Chianese R, Chioccarelli T, Ciaramella V, Fasano S, Pierantoni R, Meccariello R, Cobellis G. Cannabinoids and Reproduction: A Lasting and Intriguing History. Pharmaceuticals 3: 3275–3323, 2010.

214 215 216 217

11.

Callén L, Moreno E, Barroso-Chinea P, Moreno-Delgado D, Cortés A, Mallol J, Casadó V, Lanciego JL, Franco R, Lluis C, Canela EI, McCormick PJ. Cannabinoid receptors CB1 and CB2 form functional heteromers in brain. J Biol Chem 287: 20851–65, 2012.

218 219

12.

Cinar R, Godlewski G, Liu J, Tam J, Jourdan T, Mukhopadhyay B, HarveyWhite J, Kunos G. Hepatic cannabinoid-1 receptors mediate diet-induced insulin


resistance by increasing de novo synthesis of long-chain ceramides. Hepatology 59: 143–53, 2014.

220 221 222 223 224

13.

Despres JP, Golay A, Sjostrom L. Effects of rimonabant on metabolic risk factors in overweight patients with dyslipidemia. N Engl J Med 353: 2121–2134, 2005.

225 226 227

14.

Dossou KSS, Devkota KP, Kavanagh P V, Beutler JA, Egan JM, Moaddel R. Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay. Anal Biochem 437: 138–143, 2013.

228 229 230

15.

Elphick MR, Egertová M. The Phylogenetic Distribution and Evolutionary Origins of Endocannabinoid Signalling. In: Cannabinoids. Berlin/Heidelberg: SpringerVerlag, 2005, p. 283–297.

231 232 233

16.

Flores LE, Alzugaray ME, Cubilla MA, Raschia MA, Del Zotto HH, Román CL, Suburo ÁM, Gagliardino JJ. Islet Cannabinoid Receptors. Pancreas 42: 1085– 1092, 2013.

234 235 236 237

17.

Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O, Rössner S. Effects of the cannabinoid-1 receptor blocker rimonabant on weight reduction and cardiovascular risk factors in overweight patients: 1-year experience from the RIO-Europe study. Lancet 365: 1389–97, 2005.

238 239 240 241

18.

Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, Bouaboula M, Shire D, Fur G, Casellas P. Expression of Central and Peripheral Cannabinoid Receptors in Human Immune Tissues and Leukocyte Subpopulations. Eur J Biochem 232: 54–61, 1995.

242 243 244 245

19.

González-Mariscal I, Krzysik-Walker SM, Doyle ME, Liu Q-R, Cimbro R, Santa-Cruz Calvo S, Ghosh S, Cieśla Ł, Moaddel R, Carlson OD, Witek RP, O’Connell JF, Egan JM. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism. Sci Rep 6: 33302, 2016.

246 247 248 249 250 251

20.

González-Mariscal I, Montoro RA, Doyle ME, Liu Q-R, Rouse M, O’Connell JF, Santa-Cruz Calvo S, Krzysik-Walker SM, Ghosh S, Carlson OD, Lehrmann E, Zhang Y, Becker KG, Chia CW, Ghosh P, Egan JM. Absence of cannabinoid 1 receptor in beta cells protects against high-fat/high-sugar dietinduced beta cell dysfunction and inflammation in murine islets. Diabetologia In press;, 2018.

252 253

21.

Henstridge CM, Brown AJ, Waldhoer M. GPR55: Metabolic Help or Hindrance? Trends Endocrinol Metab 27: 606–608, 2016.

254 255 256 257 258

22.

Jourdan T, Godlewski G, Cinar R, Bertola A, Szanda G, Liu J, Tam J, Han T, Mukhopadhyay B, Skarulis MC, Ju C, Aouadi M, Czech MP, Kunos G. Activation of the Nlrp3 inflammasome in infiltrating macrophages by endocannabinoids mediates beta cell loss in type 2 diabetes. Nat Med 19: 1132– 40, 2013.

259 260 261

23.

Jourdan T, Szanda G, Cinar R, Godlewski G, Holovac DJ, Park JK, Nicoloro S, Shen Y, Liu J, Rosenberg AZ, Liu Z, Czech MP, Kunos G. Developmental Role of Macrophage Cannabinoid-1 Receptor Signaling in Type 2 Diabetes.


Diabetes 66: 994–1007, 2017.

262 263 264 265 266 267

24.

Juan-Pico P, Fuentes E, Bermudez-Silva FJ, Javier Diaz-Molina F, Ripoll C, Rodriguez de Fonseca F, Nadal A, Juan-Picó P, Bermúdez-Silva FJ, Javier Díaz-Molina F, Rodríguez de Fonseca F. Cannabinoid receptors regulate Ca(2+) signals and insulin secretion in pancreatic beta-cell. Cell Calcium 39: 155– 62, 2006.

268 269 270

25.

Kargl J, Balenga N, Parzmair GP, Brown AJ, Heinemann A, Waldhoer M. The cannabinoid receptor CB1 modulates the signaling properties of the lysophosphatidylinositol receptor GPR55. J Biol Chem 287: 44234–48, 2012.

271 272 273 274

26.

Kim W, Doyle ME, Liu Z, Lao Q, Shin Y-K, Carlson OD, Kim HS, Thomas S, Napora JK, Lee EK, Moaddel R, Wang Y, Maudsley S, Martin B, Kulkarni RN, Egan JM. Cannabinoids inhibit insulin receptor signaling in pancreatic β-cells. Diabetes 60: 1198–209, 2011.

275 276 277

27.

Kim W, Lao Q, Shin Y-K, Carlson OD, Lee EK, Gorospe M, Kulkarni RN, Egan JM. Cannabinoids induce pancreatic β-cell death by directly inhibiting insulin receptor activation. Sci Signal 5: ra23, 2012.

278 279 280

28.

Lauckner JE, Hille B, Mackie K. The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins. Proc Natl Acad Sci U S A 102: 19144–9, 2005.

281 282 283

29.

Lauckner JE, Jensen JB, Chen H-Y, Lu H-C, Hille B, Mackie K. GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current. Proc Natl Acad Sci U S A 105: 2699–704, 2008.

284 285

30.

Li C, Bowe JE, Jones PM, Persaud SJ. Expression and function of cannabinoid receptors in mouse islets. Islets 2: 293–302, 2010.

286 287 288

31.

Li C, Jones PM, Persaud SJ. Cannabinoid receptors are coupled to stimulation of insulin secretion from mouse MIN6 beta-cells. Cell Physiol Biochem 26: 187– 96, 2010.

289 290 291

32.

Li C, Vilches-Flores A, Zhao M, Amiel SA, Jones PM, Persaud SJ. Expression and function of monoacylglycerol lipase in mouse β-cells and human islets of Langerhans. Cell Physiol Biochem 30: 347–58, 2012.

292 293 294 295 296

33.

Liu J, Zhou L, Xiong K, Godlewski G, Mukhopadhyay B, Tam J, Yin S, Gao P, Shan X, Pickel J, Bataller R, O’hare J, Scherer T, Buettner C, Kunos G. Hepatic Cannabinoid Receptor-1 Mediates Diet-Induced Insulin Resistance via Inhibition of Insulin Signaling and Clearance in Mice. Gastroenterology 142: 1218–1228.e1, 2012.

297 298

34.

Lu H-C, Mackie K. An Introduction to the Endogenous Cannabinoid System. Biol Psychiatry 79: 516–525, 2016.

299 300 301 302

35.

Maccarrone M, Bab I, Bíró T, Cabral GA, Dey SK, Di Marzo V, Konje JC, Kunos G, Mechoulam R, Pacher P, Sharkey KA, Zimmer A. Endocannabinoid signaling at the periphery: 50 years after THC. Trends Pharmacol Sci 36: 277– 296, 2015.


303 304 305 306 307

36.

Malenczyk K, Girach F, Szodorai E, Storm P, Segerstolpe Å, Tortoriello G, Schnell R, Mulder J, Romanov RA, Borók E, Piscitelli F, Di Marzo V, Szabó G, Sandberg R, Kubicek S, Lubec G, Hökfelt T, Wagner L, Groop L, Harkany T. A TRPV1‐to‐secretagogin regulatory axis controls pancreatic β‐cell survival by modulating protein turnover. EMBO J 36: 2107–2125, 2017.

308 309 310 311

37.

Malenczyk K, Jazurek M, Keimpema E, Silvestri C, Janikiewicz J, Mackie K, Di Marzo V, Redowicz MJ, Harkany T, Dobrzyn A. CB1 cannabinoid receptors couple to focal adhesion kinase to control insulin release. J Biol Chem 288: 32685–99, 2013.

312 313 314 315

38.

Malenczyk K, Keimpema E, Piscitelli F, Calvigioni D, Björklund P, Mackie K, Di Marzo V, Hökfelt TGM, Dobrzyn A, Harkany T. Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture. Proc Natl Acad Sci U S A 112: E6185-94, 2015.

316 317 318 319 320

39.

Matias I, Gonthier M-P, Orlando P, Martiadis V, De Petrocellis L, Cervino C, Petrosino S, Hoareau L, Festy F, Pasquali R, Roche R, Maj M, Pagotto U, Monteleone P, Di Marzo V. Regulation, function, and dysregulation of endocannabinoids in models of adipose and beta-pancreatic cells and in obesity and hyperglycemia. J Clin Endocrinol Metab 91: 3171–80, 2006.

321 322 323 324

40.

McKillop AM, Moran BM, Abdel-Wahab YHA, Flatt PR. Evaluation of the insulin releasing and antihyperglycaemic activities of GPR55 lipid agonists using clonal beta-cells, isolated pancreatic islets and mice. Br J Pharmacol 170: 978–90, 2013.

325 326 327 328

41.

McKillop AM, Moran BM, Abdel-Wahab YHA, Gormley NM, Flatt PR. Metabolic effects of orally administered small-molecule agonists of GPR55 and GPR119 in multiple low-dose streptozotocin-induced diabetic and incretinreceptor-knockout mice. Diabetologia 59: 2674–2685, 2016.

329 330 331 332

42.

Moaddel R, Rosenberg A, Spelman K, Frazier J, Frazier C, Nocerino S, Brizzi A, Mugnaini C, Wainer IW. Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening. Anal Biochem 412: 85–91, 2011.

333 334

43.

Nakata M, Yada T. Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet β-cells via CB1 receptors. Regul Pept 145: 49–53, 2008.

335 336 337

44.

Pyszniak M, Tabarkiewicz J, Łuszczki J. Endocannabinoid system as a regulator of tumor cell malignancy – biological pathways and clinical significance. Onco Targets Ther Volume 9: 4323–4336, 2016.

338 339

45.

Reggio P. Endocannabinoid Binding to the Cannabinoid Receptors: What Is Known and What Remains Unknown. Curr Med Chem 17: 1468–1486, 2010.

340 341 342 343

46.

Romero-Zerbo SY, Rafacho A, Díaz-Arteaga A, Suárez J, Quesada I, Imbernon M, Ross RA, Dieguez C, Rodríguez de Fonseca F, Nogueiras R, Nadal A, Bermúdez-Silva FJ. A role for the putative cannabinoid receptor GPR55 in the islets of Langerhans. J Endocrinol 211: 177–85, 2011.

344

47.

Romero-Zerbo SY, Ruz-Maldonado I, Espinosa-Jiménez V, Rafacho A,


Gómez-Conde AI, Sánchez-Salido L, Cobo-Vuilleumier N, Gauthier BR, Tinahones FJ, Persaud SJ, Bermúdez-Silva FJ. The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese prediabetic mice. Sci Rep 7: 3946, 2017.

345 346 347 348 349 350 351

48.

Ryberg E, Larsson N, Sjögren S, Hjorth S, Hermansson N-O, Leonova J, Elebring T, Nilsson K, Drmota T, Greasley PJ. The orphan receptor GPR55 is a novel cannabinoid receptor. Br J Pharmacol 152: 1092–101, 2007.

352 353 354

49.

Ryberg E, Vu HK, Larsson N, Groblewski T, Hjorth S, Elebring T, Sjögren S, Greasley PJ. Identification and characterisation of a novel splice variant of the human CB1 receptor. FEBS Lett 579: 259–64, 2005.

355 356 357 358

50.

Sawzdargo M, Nguyen T, Lee DK, Lynch KR, Cheng R, Heng HH., George SR, O’Dowd BF. Identification and cloning of three novel human G proteincoupled receptor genes GPR52, ΨGPR53 and GPR55: GPR55 is extensively expressed in human brain. Mol Brain Res 64: 193–198, 1999.

359 360 361

51.

Scheen AJ, Finer N, Hollander P, Jensen MD, Van Gaal LF. Efficacy and tolerability of rimonabant in overweight or obese patients with type 2 diabetes: a randomised controlled study. Lancet 368: 1660–72, 2006.

362 363

52.

Sharkey KA, Wiley JW. The Role of the Endocannabinoid System in the Brain– Gut Axis. Gastroenterology 151: 252–266, 2016.

364 365 366

53.

Shire D, Carillon C, Kaghad M, Calandra B, Rinaldi-Carmona M, Fur G Le, Caput D, Ferrara P. An Amino-terminal Variant of the Central Cannabinoid Receptor Resulting from Alternative Splicing. J Biol Chem 270: 3726–3731, 1995.

367 368 369

54.

Starowicz KM, Cristino L, Matias I, Capasso R, Racioppi A, Izzo AA, Di Marzo V. Endocannabinoid dysregulation in the pancreas and adipose tissue of mice fed with a high-fat diet. Obesity (Silver Spring) 16: 553–65, 2008.

370 371 372

55.

Straiker A, Wager-Miller J, Hutchens J, Mackie K. Differential signalling in human cannabinoid CB1 receptors and their splice variants in autaptic hippocampal neurones. Br J Pharmacol 165: 2660–71, 2012.

373 374 375

56.

Tharp WG, Lee Y-H, Maple RL, Pratley RE. The cannabinoid CB1 receptor is expressed in pancreatic delta-cells. Biochem Biophys Res Commun 372: 595– 600, 2008.

376 377 378 379

57.

Zhang X, Gao S, Niu J, Li P, Deng J, Xu S, Wang Z, Wang W, Kong D, Li C. Cannabinoid 2 Receptor Agonist Improves Systemic Sensitivity to Insulin in HighFat Diet/Streptozotocin-Induced Diabetic Mice. Cell Physiol Biochem 40: 1175– 1185, 2016.

380 381 382

58.

Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sørgård M, Di Marzo V, Julius D, Högestätt ED. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400: 452–457, 1999.

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CONFLICT OF INTEREST

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The authors report no financial relationships with commercial interests.

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FIGURE LEGENDS

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Figure 1. The ECS. (A) Schematic of synthesis and degradation of ECs. (B) The CBRs

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are the classical CB1R, CB2R and non-classical GPR55, which are present on the

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plasma membrane. Additionally, CB1R is also present in the outer mitochondrial

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membrane in some tissues. The three CBRs are expressed in brain. In the periphery,

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CB1R and GPR55 are expressed in the tissues as shown.

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Figure 2. CB1R actions in β-cells. Activation of CB1R downregulates GLP-1R and IR

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activation, which in turn diminishes GLP-1-mediated insulin secretion, and also reduces

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Akt phosphorylation and its downstream signaling pathways. Activation of CB1R

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activates the MAPK pathway, and induces caspase 3 cleavage and expression of

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transcription factors that would lead to a metabolic shift in β-cells. Direct or indirect

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CB1R action on mitochondria in β-cells remain to be determined, but together with

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CB1R induction of ceramide synthesis, the metabolic shift induces increased

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intracellular oxidative stress. Altogether, chronic over activation of ECS in β-cells leads

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to reduced function, i.e. decreased insulin secretion, reduced proliferation, activated

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resident macrophages and reduced β-cell viability.


