CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the

modulation of GIP release and pancreatic beta cell function

A. G. McCloskey1, M. G. Miskelly1, C. B. T. McMullen1, M.A. Nesbit1, K. A. Christie2, A.

I. Owolabi1, P. R. Flatt1, A. M. McKillop1

1School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, Northern Ireland.2Center for Genomic Medicine, Massachusetts General Hospital & Harvard Medical School, 185 Cambridge St. Boston, MA 02115, USA

Short title: Effects of GPR55 agonists on insulin and GIP secretion

Word count for Abstract:

Word count for main body:

Number of references:

Number of figures: 5 (colour required for figures 1 & 3)

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Corresponding author:

Aine M. McKillop, School of Biomedical Sciences, Ulster University, Cromore Road,

Coleraine, BT52 1SA, Northern Ireland. Tel: +44 (0)28 70123066. Fax: +44(0)2870124965.

Email: am.mckillop@ulster.ac.uk

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ABSTRACT

G-protein coupled receptor-55 (GPR55), an endocannabinoid receptor, is a novel anti-diabetic

target. This study aimed to assess the metabolic functionality of GPR55 ligands using

CRISPR/Cas9 gene editing to determine their regulatory role in beta cell function and

incretin-secreting enteroendocrine cells. A clonal Gpr55 knockout beta cell line was

generated by CRISPR/Cas9 gene editing to investigate insulin secretion and Gpr55

signalling. Acute effects of GPR55 agonists were investigated in high fat fed (HFD) diabetic

HsdOla:TO (Swiss TO) mice. Atypical and endogenous endocannabinoid ligands (10-7-10-

4M) stimulated insulin secretion (p<0.05-0.001) in rodent (BRIN-BD11) and human (1.1B4)

beta cells, with 2-2.7-fold (p<0.001) increase demonstrated in BRIN-BD11 cells (10-4M). The

insulinotropic effect of Abn-CBD (42%), AM251 (30%) and PEA (53%) were impaired

(p<0.05) in Gpr55 knockout BRIN-BD11 cells, with the secretory effect of O-1602

completely abolished (p<0.001). Gpr55 ablation abolished the release of intracellular Ca2+

upon treatment with O-1602, Abn-CBD and PEA. Upregulation of insulin mRNA by Abn-

CBD and AM251 (1.7-3-fold; p<0.01) was greatly diminished (p<0.001) in Gpr55 null cells.

Orally administered Abn-CBD and AM251 (0.1µmol/kgBW) improved GIP (p<0.05-

p<0.01), GLP-1 (p<0.05-p<0.001), glucose tolerance (p<0.001) and circulating insulin

(p<0.05-p<0.001) in HFD diabetic mice. Abn-CBD in combination therapy with DPP-IV

inhibitor (Sitagliptin) resulted in greater improvement in glucose tolerance (p<0.05) and

insulin release (p<0.05). Antagonism of Gpr55 in-vivo attenuated the glucoregulatory effects

of Abn-CBD (p<0.05). Conclusively, GPR55 agonists enhance insulin, GIP and GLP-1

release, thereby promoting GPR55 agonist monotherapy and combinational therapy as a

novel approach for the treatment of type-2-diabetes.

Keywords: Cannabinoid, CRISPR/Cas9, GPR55, GIP, GLP-1, Insulin.

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1. Introduction

G-protein coupled receptor 55 (GPR55) has been identified as a novel cannabinoid receptor

[1-4]. GPR55 activation can be stimulated by plant components (phytocannabinoids),

endogenous (endocannabinoids) and small synthetic cannabinoids ligands [2, 3, 5]. The

human GPR55 gene is expressed on chromosome 2q37.1 and encodes 319 amino acid

protein, with structure presenting binding sites complementary to other cannabinoid receptors

[6]. Mouse and rat Gpr55 share 75% and 78% sequence identity with the human sequence,

respectively [3]. The GPR55 receptor is widely expressed in tissues, including the brain,

peripheral tissues, adipose tissue, endocrine-pancreas and small intestine [2,5,7,8]. Studies

have demonstrated abundant expression of GPR55 in the gastrointestinal tract of human and

rodents, with high expression in the jejunum and ileum [9]. In addition, high expression

levels were demonstrated in neurons from the myenteric plexus, indicating a regulatory role

of GPR55 in GI tract physiology, such as secretion and motility [2].

The endocannabinoid system plays a role in numerous biological processes, such as

cardiovascular, respiratory and psychological systems [3]. However, in recent years there is a

growing body of evidence demonstrating its importance in energy metabolism and glucose

homeostasis, particularly through the potentiation of GPR55 activation [4,5,10,11]. Studies

have demonstrated abundant GPR55 gene and protein expression in mouse and human islets

of Langerhans [5,8]. Furthermore, immunohistochemical analysis in rodent tissue

demonstrated that expression of Gpr55 is exclusive to insulin-secreting beta cells, with no

staining in alpha and delta cells [5]. However, one recent study reported co-localisation of

GPR55 and glucagon in alpha cells of human islets [8].

Previously, cannabinoid receptor CB1 has been associated with the regulation of several

motor functions in the GI tract, including the control of gastric emptying and esophageal

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sphincter relaxation [12]. Whilst the CB2 receptor is not linked with motility functions,

studies have demonstrated involvement upon states of intestinal inflammation [13,14]. As

GPR55 is also activated by cannabinoids, accompanied with expression throughout the GI

tract, it is likely to play a role in intestinal physiology [9,15]. At present, the role of GPR55 in

the GI tract remains poorly understood, however, receptor expression has been shown in sites

responsible for secretory processes, such as the enteric nervous system and colon mucosa

[16,17]. Interestingly, global deletion of Gipr attenuated the glucose lowering effect of Gpr55

agonism in mice [18]. Subsequently, due to the known glucoregulatory function and abundant

expression of GPR55 throughout the GI tract, further investigation is clearly warranted to

explore the role of cannabinoids in incretin hormone release.

Upon activation, GPR55 primarily couples to Gα12/13 and Gαq intracellular G-protein

subunits, resulting in phospholipase C (PLC) and RhoA signalling, which stimulate an array

of downstream cellular responses, such as myosin contractility, cell cycle maintenance,

cellular morphological polarization, cellular secretion, cellular development and

transcriptional control [19-21]. The mechanism of GPR55-mediated insulin secretion remains

uncertain but initial studies have shown a range of endogenous and synthetic GPR55 agonists

to augment intercellular Ca2+ release, suggesting involvement of effects of inositol

trisphosphate (IP3) on intracellular calcium stores through PLCβ signalling [5,8,11].

Along with high expression of GPR55 in pancreatic beta cells, activation of the GPR55

receptor by endogenous and synthetic agonists, augments insulin secretion [5,18]. Recent

studies utilising cannabinoid agonists demonstrated the insulinotropic property of Gpr55 in

clonal BRIN-BD11 cells and human islets, potentiated through intracellular calcium mediated

signalling [5, 8]. The insulinotropic effect of the GPR55 agonists was attenuated with a

GPR55 antagonist [5]. Chronic administration of abnormal cannabidiol (Abn-CBD) improved

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glucose control and increased plasma insulin in streptozotocin-induced diabetic mice and

reduced plasma cholesterol, bodyweight and food intake, whilst marked improvements of

insulin sensitivity and glucose tolerance were observed [18].

Studies evaluating the effects of global receptor deletion using Gpr55 knockout mice reported

a mild impairment in glucose homeostasis, with no effect on glucose stimulated insulin

secretion (GSIS) [3]. Another study using isolated islets from wild type and Gpr55 knockout

mice reported that the synthetic agonist O-1602 augmented intracellular Ca2+ and insulin

secretion, with effects diminished in Gpr55 deficient islets, thus demonstrating that atypical

cannabinoids have a direct effect in the pancreatic islet through Gpr55-dependent signalling

[8]. Abn-CBD was shown to have Gpr55-dependent and independent effects in murine islets

[22]. However, use of tissues from knockout mice is greatly complicated by induction of

complementary compensatory pathways [23]. At present, specificity of endogenous

endocannabinoids and synthetic cannabinoid ligands for GPR55 is unclear, with the

possibility that many also target other novel endocannabinoid receptors, GPR18 and GPR119

[24, 25]. Even though GPR18 and GPR119 are linked with the endocannabinoid system, they

are still considered orphan receptors as their respective endogenous agonists remain unknown

[26].

The present study, for the first time has investigated the specificity and downstream

signalling of putative GPR55 agonists using wild type and Gpr55 knockout beta cells

generated by CRISPR/Cas9 gene editing. Acute metabolic effects of Gpr55 activation,

including GIP and GLP-1 release, were evaluated as a novel therapeutic approach for the

treatment of type 2 diabetes.

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2 Materials and methods:

2.1 Constructs

The S.pyogenes Cas9 vector plasmid used for the dual luciferase assay was pSpCas9(BB)-

2A-Puro (PX459) V2.0, and the S. pyogenes Cas9 vector plasmid used for FACS sorting was

pSpCas9(BB)-2A-GFP (PX458), both plasmids were a gift from Feng Zhang to author CBT

Moore (Broad Institute, MIT). An sgRNA specific to Gpr55 was designed incorporating the

start codon at position 5-8 in the guide sequence, hereafter named sgGPR55. Guide

sequences were cloned into the respective plasmids following a published protocol by Ran et

al. [27];  briefly, pSpCas9(BB)-2A-Puro and pSpCas9(BB)-2A-GFP were digested with BbsI

(NEB) and the following oligos, 5′-CACCGGAACATGAGTCAGCTAGACA-3′ and 5′-

AAACCTTGTACTCAGTCGATCTGTC-3′ (Life Technologies, UK), were annealed and

ligated into the digested plasmids. To assess Cas9 cleavage efficiency directed by sgGPR55,

a firefly luciferase expression plasmid with a 50bp insert of Gpr55 sequence encoding the

sgGPR55 target site was inserted into psiTEST-LUC-Target (York Bioscience Ltd, York,

UK), hereafter named as GPR55WT-Luc. An expression construct for Renilla luciferase

(pRL-CMV, Promega, Southampton, UK) was used for the dual-luciferase assay to normalize

transfection efficiency.

The sgRNA sequence was designed to ensure optimum cutting efficiency and specificity.

Following a CRISPR guide scoring system devised by Doench et al. 2016 (On-target score)

and Hsu et al. 2013 (Off-target score), the CRISPR guides chosen returned the best possible

score for selectively targeting a genomic region nearby GPR55 start codon

2.2 Dual-luciferase assay

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A dual-luciferase assay was used to determine cleavage efficiency of the Cas9-sgGPR55

expression construct, using methods previously described [28-30]. HEK AD293 cells (Life

Technologies, UK) were transfected using Lipofectamine 3000 (Life Technologies, UK); the

GPR55WT-Luc expression construct was co-transfected with either sgNSC or sgGPR55

constructs and a Renilla luciferase expression plasmid. Cells were incubated for 72h before

being lysed and the activities of both Firefly and Renilla luciferase quantified.

2.3 Construct transfection and FACS sorting

Wild type insulin secreting BRIN-BD11 cells were transfected with the plasmid co-

expressing Cas9, sgGPR55 and GFP using Lipofectamine 3000 following manufactures

recommended protocol. Generation, characteristics and culture of these cells have been

previously described [31]. Post-transfection 72h, cells were detached with trypsin/EDTA and

re-suspended in sterile PBS. Flow cytometry was employed to detect and FACS sort the cells

expressing GFP. Cells emitting the top 1% of FITC-A GFP expression were isolated into

single cell populations and allowed to expand under normal culture conditions for 3 weeks to

form colonies.

2.4 Sequencing and determination of Gpr55 knockout

Upon clonal expansion, gDNA was extracted using DNA extraction kit (Qiagen, UK).

Individual samples were then subjected to PCR amplification using the following primers to

amplify the targeted region: 5′-AGCCCCTCGTTCTGTGTTTA-3′ and 5′-

AGGAGGAAGGTGGGAATGTG-3′. PCR products were gel purified and ligated into the

CloneJet cloning vector (Life Technologies, UK), then transformed into competent DH5α

cells (Life Technologies, UK). DNA from 40 BRIN-BD11 cell clones was prepared using a

plasmid miniprep kit (Life Technologies, UK) following manufacturer’s protocol. Isolated

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DNA was then sequenced (Department of Biochemistry, University of Cambridge) using the

forward PCR amplification primer.

2.5 Insulin secretion from BRIN-BD11 and 1.1B4 cells:

Wild type/ Gpr55 knockout BRIN-BD11 cells and human 1.1B4 cells were used. In brief,

cells were seeded (150,000 cells per well) into 24-well plates and allowed to attach overnight.

Following a 40min pre-incubation (1.1mmol/l glucose), cells were incubated (20min; 37oC)

in KRB buffer with established stimulators of beta cell function and GPR55 agonists (10−7-

10−4mol/l). Supernatants were removed, then frozen at -20∘C until determination of insulin by

radioimmunoassay [32].

2.6 Gene expression analysis by qPCR:

mRNA was extracted from wild type and Gpr55 knockout BRIN-BD11 cells following

exposure to agonist treatment for 4h using the RNeasy Mini kit adhering to manufacturer’s

protocol (Qiagen, UK). Isolated mRNA (3µg) was converted to cDNA using SuperScript II

Reserve Transcriptase (Life Technologies, UK). SYBR green amplification parameters for

Gpr55, Glp1r and Gipr were set at 95oC for denaturation, 58oC for primer annealing and 72oC

for elongation for a total of 40 cycles, followed with melting curve analysis, with temperature

range set at 60oC to 90oC. Values were analysed using the Livak method and normalised to

Gapdh expression.

2.7 Intracellular Ca2+ and cAMP:

For intracellular Ca2+ measurement, monolayers of BRIN-BD11 cells were seeded overnight,

at a density of 80,000 cells per well in a 96-well black walled clear bottom plate. Cells were

washed with 100 μL of Krebs buffer and incubated for 1 h with Flex calcium assay kit

reagent (Promega; Madison, WI, USA) at 37°C. O-1602, PEA, OEA, AM-251 and Abn-CBD

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at 10−4 mol·L−1 were added at 5.6 mmol·L−1 glucose. Fluorometric data were obtained using

the FLEX Station scanner and test solutions were transferred using fluid transfer workstation

at a wavelength of 525 nm (Molecular Devices, Sunnyvale, CA, USA). For cAMP

determination, BRIN-BD11 cells were seeded in a 96-well plate at a density of 30,000 cells

per well. Cells were washed with 300 μL Krebs buffer for 40 min and 150 μL of GPR55

agonists at 10−4 mol·L−1 were tested at 5.6 mol·L−1 glucose. After 20 min, test solutions were

removed and 0.1 M HCL (150 μL) was added to lyse the cells. Total cAMP production in the

cell, supernatants were measured using cAMP enzyme immunoassay kit according to the

manufacturer’s protocol (Sigma-Aldrich, Poole, UK).

2.8 Acute effects of GPR55 agonists in-vivo:

All animal experiments were carried out in accordance with the UK Animal (Scientific

Procedures) Act 1986. Male lean and HFD HsdOla:TO (Swiss TO) mice (Harlan UK, 10-12

weeks old) were individually housed in an air-conditioned room at 22±2oC with a 12-h light:

12-h dark cycle and placed on a normal or high fat diet (45% fat, 20% protein, 35%

carbohydrate; percent of total energy 26.15 kJ/g; Dietex International Ltd., Witham, UK) for

20 weeks prior to experimentation. Drinking water and either normal or high fat maintenance

diet (Trouw Nutrition, Cheshire, UK) were supplied at libitum. Another group of mice was

maintained on standard rodent diet (10% fat, 30% protein, 60% carbohydrate; percent of total

energy 12.99 kJ/g, Trouw Nutrition, Cheshire, UK) and used as a model of normal controls.

18 h fasted, age-matched, fasted mice (n=6) received an oral administration (150µl) of

glucose alone (18 mmol/kg body weight) or glucose in combination with GPR55 agonists

(0.1μmol/kg body weight). Glucose and GPR55 agonists were prepared in 0.9% saline. Blood

samples (125µl) were obtained from the cut tip of the tail vein of conscious mice at the times

indicated. Plasma was separated by centrifugation at 16,060× g for 3min at 4oC and stored at

-20oC until analysis. Plasma glucose was measured using an automated glucose oxidase

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procedure with a Beckman glucose analyser (Beckman-Coulter, High Wycome, UK) and

insulin determined by radioimmunoassay [32]. Intestinal hormone secretion were determined

using specific ELISA kits; total GLP-1 (Millipore) and total GIP (Millipore). DPP-IV activity

was assessed by Gly-Pro-AMC cleavage [33].

2.9 Statistical Analysis

All data was analysed with Prism (v.5.0, GraphPad Software Inc. CA, USA) and expressed as

mean ± S.E.M. Results were compared using the Student’s t-test (non-parametric, with two-

tailed P values and 95% confidence interval) for firefly luciferase assay, or two-way analysis

of variance (ANOVA) followed by the Bonferroni post-hoc test for insulin secretion, Ca2+,

cAMP and glucose tolerance analysis. Area under the curve (AUC) was calculated using

trapezoidal rule with baseline correction. Differences in data were considered to be

statistically significant for p<0.05.

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3 Results

3.1 Development of a Gpr55 null BRIN-BD11 cell line using CRISPR/Cas9 gene editing.

The CRISPR/Cas9 construct (px458) used co-expresses GFP which was utilised to select the

cells which had been successfully transfected (Figure 1A). A sgRNA was designed to target

1bp upstream of the Gpr55 start codon to induce gene disruption by non-homologous end

joining (NHEJ); the sgRNA was targeted early in the transcript, encompassing the ATG start

codon at position 5-8 in the guide sequence and is referred to as sgGPR55. (Figure 1A,B).

sgGPR55 was shown to have good cleavage efficiency, resulting in a 60% reduction in firefly

luciferase activity (p<0.001) when assessed with a dual luciferase assay (Figure 1C). 72h

post-transfection with the Cas9-sgGPR55-GFP expression construct, cells emitting the top

1% of GFP expression were single cell sorted by FACS, followed by clonal expansion

(Figure 1D3).

To determine which cells had bi-alleleic indels, individual PCR products from clonal cell

populations were sequenced; clone 2 sequence analysis revealed a 29 and 33 nucleotide

deletion on allele 1 and allele 2 respectively (Figure 1E,F). A 2 nucleotide insertion mutation

was present on allele 2 (Figure 1E,F), indicating a bi-allelic deletion of the Gpr55 start codon

was present in this cell line that would be predicted to abolish expression of Gpr55 protein.

(Figure 1F). Gpr55 mRNA expression was ablated in Gpr55 knockout BRIN-BD11 cells,

compared to wild type (Figure 1G). Incretin receptor mRNA expression (Glp1r and Gipr)

were unaffected in the Gpr55 knockout cell line, compared to wild type cells (Figure 1G).

3.2 Effects of GPR55 agonists on insulin secretion from wild type/Gpr55 knockout

BRIN-BD11 cells and human 1.1B4 cells

The insulinotropic capabilities and specificity of a range of putative GPR55 agonists were

assessed using wild type, Gpr55 knockout BRIN-BD11 cells and human 1.1B4 cells. At

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16.7mM glucose, a synthetic CBD analogue (Abn-CBD) augmented insulin secretion by 0.8-

2.7 fold (p<0.05-0.001) at 10-7-10-4M in wild type BRIN-BD11 cells (Figure 2A). The

secretory response of Abn-CBD was significantly impaired at 10 -5-10-4M (p<0.05) in Gpr55

knockout cells, with a 42% (p<0.05) reduction demonstrated at the highest agonist

concentration (10-4M) (Figure 2A).

Synthetic CBD analogue (O-1602) induced insulin secretion at by 0.6-2.7 fold (p<0.05-

0.001) 10-7-10-4M in wild type cells (Figure 2B). The insulinotropic effect of O-1602 was

abolished at all concentrations tested (10-7-10-4M) in Gpr55 knockout cells (Figure 2B).

Rimonabant analogue (AM251) stimulated insulin secretion by 0.3-2.1 fold (p<0.05-0.001) at

10-7-10-4M in wild type cells (Figure 2C). AM251-induced insulin secretion was reduced by

30% in Gpr55 knockout cells (Figure 2C). Endogenous endocannabinoids PEA and OEA

augmented insulin secretion by 0.6-2.0 fold (p<0.001) and 0.7-2.2 fold (p<0.01-0.001) at 10-

7-10-4M, respectively in wild type cells (Figure 2D,E). The insulinotropic response of PEA

was impaired by 53% (p<0.05) at 10-4M in Gpr55 knockout cells, whilst OEA-induced

insulin secretion was retained in Gpr55 knockout cells. Complementary analysis

demonstrated that Abn-CBD and AM251 at 10-7-10-4M augmented insulin release by 1.4-2

fold (p<0.01-0.001) in human 1.1B4 cells (Figure 2F,G).

3.3 Effects of GPR55 agonists on insulin mRNA expression in wild type and Gpr55

knockout BRIN-BD11 cells.

Culture of cells with atypical cannabinoid GPR55 agonists Abn-CBD and AM251 induced a

2.8-3.0 fold (p<0.05-0.01) increase of insulin mRNA expression in wild type BRIN-BD11

cells at 5.6mM glucose, and a 1.8-2.1-fold increase (p<0.05) at 16.7mM glucose (Figure

2H,I). In contrast, Abn-CBD and AM251 was diminished (p<0.001) insulin mRNA

expression in Gpr55 KO cells at both 5.6mM and 16.7mM glucose (Figure 2J,K).

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3.4 Effects of GPR55 agonists on intracellular Ca2+ and cAMP in wild type and Gpr55

knockout BRIN-BD11 cells

Exposure of wild type BRIN-BD11 cells to AM251, PEA, O-1602 and Abn-CBD at 10 -4M

induced a prominent increase in intracellular Ca2+ (p<0.05-0.001) (Figure 3A,C). Using AUC

data, AM251 demonstrated the most potent effect, with a 17-fold increase observed,

compared to 5.6mM glucose alone (Figure 3C). Increases of intracellular Ca2+ mediated by

PEA, O-1602 and Abn-CBD were abolished when assessed in Gpr55 knockout cells, whilst

the effect of AM251 was reduced by 43% (Figure 3B, D). None of the agonists affected

cAMP production in either wild type nor Gpr55 knockout cells (Figure 3E,F).

3.5 Effects of GPR55 agonists on glucose tolerance and secretion of insulin and incretin

hormones in high fat fed diabetic mice.

An oral glucose tolerance test (OGTT) was performed to assess the anti-diabetic capabilities

of Abn-CBD and AM251 in fasted, HFD mice. Abn-CBD and AM251 were assessed in

monotherapy and combination therapy (Sitagliptin), whilst GPR55 antagonist CBD was used

to assess agonist specificity. Oral administration of Abn-CBD and AM251 improved the

glucose excursion (p<0.05-0.001) (Figure 4A,C), with AUC data showing Abn-CBD and

AM251 to decrease plasma glucose by 28% (p<0.01) and 31% (p<0.01) respectively (Figure

4E,G). Abn-CBD in combination with Sitagliptin further improved glucose tolerance by 24%

(p<0.05), compared to agonist monotherapy. Antagonising Gpr55 with CBD impaired the

glucose lowering capabilities of Abn-CBD by 36% (p<0.05) (Figure 4A).

Abn-CBD (p<0.05) and AM251 (p<0.001) increased plasma insulin concentrations when

administered with glucose (Figure 4B,D). The responses were enhanced by 25% and 60%,

respectively, when assessed with AUC data (Figure 4F,H). Abn-CBD in combination with

Sitagliptin demonstrated an additive 20% insulinotropic effect (p<0.05), compared to agonist

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monotherapy (Figure 4F). Antagonising Gpr55 with CBD impaired the insulinotropic

response of Abn-CBD by 36% and abolished the response of AM251 (p<0.001).

HFD mice treated with Abn-CBD had increased plasma GIP (p<0.05) and GLP-1 (p<0.001).

AM-251 administered in vivo also increased plasma GIP (p<0.05) and GLP-1 (p<0.05)

release (Figure 5A,B,D,E). DPP-IV activity was reduced by Sitagliptin (p<0.001) in high fat

fed mice (Figure 5C,F). Abn-CBD and AM251 when administered alone had no effect on

DPP-IV activity in vivo, however in combination with Sitagliptin resulted in a reduction in

DPP-IV activity (p<0.001) (Figure 5C,F).

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4 Discussion

Novel therapies that enhance beta cell function and modulate incretin secretion are required

for the treatment of type 2 diabetes. GPCRs are expressed in a variety of tissues and several

GPCRs have been identified on enteroendocrine cells in the gastrointestinal tract and shown

to partly moderate plasma glucose and insulin release by activation of the incretin system and

secretion of GIP [15]. In recent years, interest has intensified towards the involvement of the

endocannabinoid system in the maintenance of glucose homeostasis through GPCR signalling

with long chain fatty acid agonists [3, 4]. In addition, endocannabinoid ligands have been

shown to display a range of beneficial effects on metabolic control, with CB1 inverse agonist

Rimonabant (SR141716) once used as an anti-obesity drug prior to its withdrawal due to

mood altering side effects [34].

Although the function of the CB1 and CB2 receptors are well characterised within the

endocannabinoid system, the role of the recently discovered endocannabinoid receptors

GPR55, GPR119 and GPR18 remains poorly understood [24, 25, 26]. However, GPR119 has

been shown to potentiate GIP release upon selective agonism with AR231453 in mice [35].

Previously, studies found that CB1 activation inhibited secretion of GIP from intestinal K

cells in rodents through the activation of inhibitory Gα i-coupled signalling [36]. In contrast,

GPR55 activates stimulatory downstream signalling events (Gαq and Gα12/13) and shares

limited sequence homology with the CB1 and CB2 receptors, and therefore has been

hypothesised to be responsible for the non-CB1/CB2 effects of endocannabinoid ligands [3].

GPR55 is a rhodopsin (class A) GPCR that has been previously shown to demonstrate anti-

diabetic effects through the regulation of insulin secretion and glycaemic control [5, 19].

Studies have demonstrated abundant expression of GPR55 in the gastrointestinal tract of

human, with high expression in the jejunum and ileum [9]. In the present study, the acute

metabolic effects and specificity of several naturally occurring and atypical endocannabinoid

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ligands were assessed using HFD mice and a clonal Gpr55 knockout cell line, developed

using CRISPR/Cas9 gene editing. Moreover, for the first time, the effects of Gpr55 agonism

towards GIP and GLP-1 release were investigated with agonist combinational therapy

(Sitagliptin) evaluated as a novel therapeutic approach. Sitagliptin was utilised in

combination to prolong the bioactivity of endogenously released incretins upon Gpr55

agonism

To establish the Gpr55 knockout BRIN-BD11 cells, the Cas9 enzyme was directed to cleave

immediately adjacent to the Gpr55 start codon, stimulating the NHEJ DNA repair machinery

which often, as in the selected clone, introduces indel mutations at the cleavage site. The

resultant Gpr55 knockout clone contained a bi-allelic deletion of the Gpr55 start codon, with

Gpr55 mRNA expression abolished.

Subsequently, the insulin secretory effects and specificity of putative GPR55 agonists were

assessed using the wild type and Gpr55 knockout BRIN-BD11 cells. Naturally occurring

(OEA, PEA) and atypical (Abn-CBD, AM251, O-1602) agonists demonstrated potent

insulinotropic effects in wild type cells. The insulin secretory effect of CBD analogue (Abn-

CBD) was impaired in the knockout cell line by 42%, indicating the involvement of Gpr55 in

Abn-CBD mediated insulin secretion. In addition, the secretory response of another CBD

analogue (O-1602) was totally abolished in the knockout cell line, suggesting O-1602 as a

selective agonist for Gpr55 activation in the pancreatic beta cell.

Rimonabant analogue (AM251) demonstrated insulinotropic capabilities in wild type and

Gpr55 knockout BRIN-BD11 cells. However, the secretory response of AM251 was reduced

by 30% in the knockout cell line, indicating that AM251 induced insulin secretion is only

partly driven by Gpr55 in pancreatic beta cells.

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Endogenous cannabinoid ligands (OEA, PEA) augmented insulin secretion in wild type and

Gpr55 knockout BRIN-BD11 cells, illustrating the non-selective properties of the

endogenous endocannabinoids. The secretory response of OEA was fully retained within the

knockout pancreatic cell line, thus, implicating the involvement of an alternate cannabinoid

receptor (e.g. GPR119) for OEA-induced insulin release [24]. In contrast, the secretory

response of PEA was impaired by 53% in the Gpr55 deficient cell line, demonstrating that

PEA-induced insulin secretion is partially mediated through Gpr55 with potential synergistic

agonisation with another endocannabinoid receptor, such as Gpr119 and Gpr18 [24].

Complementary gene expression analysis by qPCR demonstrated increased insulin mRNA

expression upon Abn-CBD and AM251 treatment in normoglycaemic and hyperglycaemic

conditions; indicating the role of the cannabinoid ligands in insulin biosynthesis.

Interestingly, Abn-CBD and AM251 treatment greatly diminished insulin mRNA expression

in Gpr55 KO cells, thus, revealing the importance of Gpr55 in the production of intracellular

insulin stores.

Intracellular Ca2+ and cAMP signalling studies were employed on wild type and Gpr55

knockout pancreatic cells to determine downstream effects of the endocannabinoid ligands.

Complementary to insulin secretory studies, increases of intracellular Ca2+ observed in wild

type cells by O-1602, PEA and Abn-CBD, were abolished in Gpr55 knockout cells, while

response to AM251 was only impaired. No changes in cAMP production were observed in

wild type or knockout cells, suggesting Gpr55 signalling is primarily modulated through Gαq

activation and release of Ca2+ from intracellular stores [37]. However, Gα12/13 activation

may also contribute to the actions of Gpr55 activation.

Evaluation of acute effects of potent GPR55 agonists Abn-CBD and AM251 on glucose

tolerance in HFD mice demonstrated strong glucose lowering and insulinotropic activities. In

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addition, oral administration of both GPR55 agonists stimulated GIP and GLP-1 secretion,

revealing the role of Gpr55 in enteroendocrine cell function for the first time. The mechanism

of incretin release is not fully understood as both direct and indirect signalling pathways may

contribute to the effects observed. GPR55 expression has been previously reported in the

enteric nervous system and in the brainstem, which houses the dorsal motor nucleus of the

vagal nerve [16]. GPR55-mediated incretin release may be partly driven by neurological

signalling, however, further studies are warranted to reveal the exact mechanism of action.

The effect of GPR55 agonist combinational therapy was studied using the DPP-IV inhibitor

Sitagliptin to prolong the half-life of the incretin hormones. As anticipated, Abn-CBD and

AM251 exhibited enhanced glucose lowering and insulinotropic capabilities with DPP-IV

inhibition, by stimulating incretin (GIP, GLP-1) secretion through Gpr55 activation,

supplemented with prolonged incretin action [38]. Antagonising Gpr55 with CBD impaired

the glucose lowering and insulin secretory capabilities of Abn-CBD and AM251, which

demonstrates the specificity of the GPR55 ligands in the diabetic animal model.

In conclusion, the specificity of endocannabinoid ligands towards Gpr55 was evaluated with

O-1602, Abn-CBD, AM251 and PEA demonstrating Gpr55-dependent insulin secretion

through intracellular Ca2+ modulation. In addition, the insulinotropic actions of GPR55

ligands was also demonstrated in clonal human beta cells. The biological activation of Gpr55

with Abn-CBD and AM251 demonstrated strong glucose lowering and insulinotropic

capabilities in HFD diabetic mice, with enhanced secretion of GIP and GLP-1. Agonist

combinational therapy using Sitagliptin enhanced the glucose lowering capabilities of the

endocannabinoid ligands, demonstrating a novel therapeutic approach for the maintenance of

glucose homeostasis in the treatment of type 2 diabetes.

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Acknowledgement

These studies were supported by Diabetes UK. AMC contributed to the data curation, formal

analysis, methodology, project admin and writing. AMK contributed to the conceptualization,

formal analysis, funding acquisition, investigation, methodology, project admin, supervision

and writing. PRF contributed to the funding acquisition, methodology, supervision and

writing. MGM, CBTM, KC, AIO and MAN contributed to the formal analysis, investigation

and editing.

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Figure legends

Figure 1. Specific targeting and genomic deletion of Gpr55 in BRIN-BD11 cells using

CRISPR/Cas9 gene editing. (A) Schematic of px458 construct used. (B) sgRNA and PAM

sequences (underlined) and predicted genomic DNA cleavage sites (indicated by arrow)

encoding Gpr55 exon 1. (C) Dual luciferase assay demonstrating sgGPR55 cutting efficiency

compared to non-specific control (sgNSC). (D) Phase contrast photomicrograph and sorting

gate of GFP+ cells 72 h post-lipofection with sgGPR55 construct prior to fluorescence-

activated cell sorting (FACS). (E) Single allele DNA sequencing traces of Gpr55 knockout

clone. (F) Bi-allelic genomic deletion of the Gpr55 start codon [circled]. (G) Gpr55, Gipr

and Glp1r mRNA expression by qPCR. Values are mean ± SEM (C: n=8, G: n=3).

Figure 2. Insulinotropic effects of GPR55 agonists in clonal beta cells. (A-E) secretory effect

at 16.7mM glucose in wild type and Gpr55 knockout clonal BRIN-BD11 cells. (F-G)

secretory effects in human 1.1B4 cells. qPCR analysis demonstrating insulin mRNA

expression in wild type (H, I) and Gpr55 knockout (J, K) BRIN-BD11 cells upon agonist

treatment (10-4M) for 4 h at 5.6mM and 16.7mM glucose. Values are mean ± SEM (A-G;

n=8, H-K; n=3). * p<0.05, ** p<0.01, *** p<0.001, compared to respective glucose control.

+ p<0.05, ++ p<0.01, +++ p<0.001, compared to wild type cells.

Figure 3. Effects of GPR55 agonists on intracellular Ca2+ (A-D) and cAMP (E-F) in wild

type (A, C, E) and Gpr55 knockout (B, D, F) BRIN-BD11 cells. Values are mean ± SEM

(n=6). * p<0.05, ** p<0.01, *** p<0.001, compared to glucose control.

Figure 4. Effects of GPR55 agonist (Abn-CBD and AM251) monotherapy and combination

therapies on plasma glucose excursion (A, C) and insulin (B, D). AUC data for Abn-CBD (E,

F) and AM251 (G, H) are also shown. Glucose (18mmol/kg bw) was administered orally to

HFD mice in combination with GPR55 agonists Abn-CBD/AM251 (0.1µmol/kg bw), GPR55

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antagonist CBD (0.1µmol/kg bw) and Sitagliptin (50mg/kg bw). Values are presented as

mean ± SEM (n = 6). * p<0.05, ** p<0.01, *** p<0.001, compared to HFD glucose control.

Δp<0.05 and ΔΔΔp<0.001, compared to agonist monotherapy.

Figure 5. Effects of GPR55 agonists Abn-CBD and AM251 on circulating total GLP-1 (A,

D), total GIP (B, E) and DPP-IV activity (C, F). Glucose (18mmol/kg bw) was administered

orally to HFD mice in combination with GPR55 agonists Abn-CBD/AM251 (0.1µmol/kg

bw) and Sitagliptin (50mg/kg bw). Values are presented as mean ± SEM (n = 6). *p<0.05, **

p<0.01, *** p<0.001, compared to HFD glucose control.

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Figure 1

W.T: CATCTACTGTTGGTGTTGCAGGCCAGAACATGAGTCAGCTAGACAGTAACAACTGCTCGTTCGTTTT

Allele 1: CATCTACTGTTGGTGTTGCA-----------------------------CAACTGCTCGTTCGTTTT

Allele 2: CATCTACTGTTGGTGTTGCTC-------------------------------CTGCTCGTTCGTTTT

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Figure 3.

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