British J Pharmacology - 2024 - Wang - Differential contributions of G protein‐ or arrestin subtype‐mediated signalling-html.html

; Rodriguez-Diaz

et al.,

2011

; van der Meulen et al.,

). Besides the

cell loop

and

cell loop, many other cell

–

cell loops, such as macrophage-

cell loop in mouse diabetes models, play important roles in maintain-

ing glucose homeostasis (Golden & Simmons,

; Ying et al.,

For instance, the insulin peptide fragment, insB:9-23, promoted islet-

resident macrophage proliferation via an OLFR109 dependent

cell-

macrophage circuit and a

-arrestin1-mediated

CCL2

pathway (Cheng

et al.,

). The islet amyloid polypeptide (IAPP), which is secreted

by islet

cell, stimulates islet-resident macrophages to produce

IL-1

and inhibits glucose-stimulated insulin secretion (Arnush et al.,

1998

;

Masters et al.,

2010

). These studies indicated the importance of com-

munication circuits among different cell types within islets in main-

taining glucose homeostasis. Not only protein or peptides, but fatty

acids also participate in signalling circuits within islets. For example,

our recent study found that biased endogenous lipid-GPR120 signal-

ling modulates pancreatic glucose homeostasis to different extents,

and the structures of GPR120 with the two endogenous ligands oleic

acid (OA) and linoleic acid (LA) provided important mechanisms of the

biased signalling of GPR120 at the atomic resolution level (Du

et al.,

; Mao et al.,

). Notably,

UCN3

secreted by pancreatic

cells is to induce somatostatin secretion from pancreatic

cells via

the activation of the

CRF

receptor

, a 7TM receptor (this signal axis

can be abbreviated as UCN3-CRF

receptor-SST). Importantly, the

essential role of the normal UCN3-CRF

receptor-SST signalling axis

in maintaining glucose homeostasis and diabetes prevention has been

recently revealed (Li et al.,

; van der Meulen et al.,

UCN3

(urocortin 3) is a peptide hormone that is abundantly dis-

tributed in the central nervous system (e.g., hypothalamus) and

peripheral tissues, highly expressed in the digestive tract, liver,

What is already known?

•

Islet cell circuits function via crosstalk and paracrine sig-

nalling to regulate blood glucose levels.

•

UCN3 secreted by pancreatic

cells induces somato-

statin secretion via activation of CRF

receptors.

What does this study add?

•

Coupling of Gs, G

and

-arrestin1 to CRF

receptors all

contribute to UCN3-induced SST secretion.

•

Different UCN3 levels have distinct effects on glucose

homeostasis,

depending

different

downstream

effectors.

What is the clinical significance?

•

Our findings provide a signal transduction mechanism to

target CRF

receptors in diabetes.

WANG

ET AL

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pancreas and gonads, and expressed specifically by

cells in both

mouse and human islets. The widespread expression of UCN3 in

different tissues of animals suggests that UCN3 may be involved in

a variety of physiological functions, such as the contraction/

relaxation of myometrial cells and metabolic regulation (Blank

et al.,

2003

; Harlé et al.,

; Jamieson et al.,

2011

; Karteris

et al.,

2004

; Xiong et al.,

) (Figure

). The presence of both

UCN3 and its receptor in pancreatic islets suggests that UCN3 may

be a local factor regulating islet function, such as hormone secre-

tion. There is evidence that UCN3 is co-secreted with insulin upon

glucose sensing and promotes somatostatin secretion from

cells

(Li et al.,

2007

; Li et al.,

; van der Meulen et al.,

Although UCN3-CRF

receptor-SST is the central axis in the

cell

loop in pancreatic islets and plays essential roles in maintaining glu-

cose

homeostasis,

two

important

questions

have

not

been

addressed: (1) since the CRF

receptor is known to activate multiple

protein

subtypes

and

arrestins

(Ma

al.,

;

Zhao

et al.,

), how does the signalling mediated by these different

effectors (G protein or arrestin subtypes) contribute to the paracrine

signalling of the

cell loop and UCN3-induced SST secretion via

the activation of the CRF

receptor? (2) Since UCN3 is present at

different concentrations under physiological or pathological condi-

tions, whether different UCN3 concentrations have distinct effects

on glucose homeostasis? These questions are interesting because

the elucidation of functional outcomes and mechanistic details of

biased signalling downstream of a particular GPCR may have thera-

peutic values for disease treatment and help us to understand the

complexity of intrcellular events of a specific physiological process.

We speculated that not only particular ligands, but also certain

endogenous ligands of a particular GPCR, such as UCN3, may be

able to activate multiple G protein subtypes (Gs, G

) and

-arrestin1, with potentially different EC50 and Emax values. This

knowledge could be helpful for our understanding of the paracrine

network between the pancreatic

cells and

cells.

M E T H O D S

2.1

Animals

Sst-Cre

mice (stock number 013044) with a C57BL/6 background

were obtained from the Jackson Laboratory (Bal Harbor, USA) and

were used in our previous studies (Du et al.,

; Li et al.,

fl/fl

Arrb1

and

Arrb2

mice on a C57BL/6 background

were obtained from Dr R.J. Lefkowitz (Duke University, Durham, NC,

USA) and G. Pei (Tongji University, Shanghai, China) and maintained

and crossed as described previously (Du et al.,

; Liu et al.,

The

Rosa

lsl-RFP

mice were generated by introducing loxP-STOP-loxP

and TdTomato fragments at the Rosa26 locus. The STOP sequence

between two loxp sites could be cut by Cre recombinase to initiate

the expression of RFP. We crossed

Rosa

lsl-RFP

mice with

Sst-Cre

mice

and then generated

Sst-Cre

Rosa

lsl-RFP

mice.

Rosa

lsl-RFP

mice were

obtained from Tian Xue (Do et al.,

2009

; Li et al.,

). Non-obese

diabetic/LtJ (NOD) mice (Stock No.13008A) were purchased from

Beijing HFK Bioscience. All animal care and experiments were

reviewed and approved by the Animal Use Committee of Shandong

University Cheeloo College of Medicine. Animal studies are reported

in compliance with the ARRIVE guidelines (Percie du Sert et al.,

)

and with the recommendations made by the

British Journal of Pharma-

cology

(Lilley et al.,

2.2

Diabetic mouse model induced by low dose

streptozocin (STZ)

A diabetic mouse model was induced by low dose STZ according to

our previously described method (Cheng et al.,

; Du et al.,

STZ mice model were obtained by intraperitoneal injection of strepto-

zocin (20 mg

day

) (STZ Solarbio Cat. S8050, Beijing, China) for

5 days. After modeling for 5 days, it is preferable to test the fasting

blood glucose level immediately (or no more than 72 h) to confirm

that it is greater than 15 mmol

, indicating a successful modeling.

2.3

RNA extraction and quantitative reverse-

transcriptase PCR

RNA extraction and Quantitative reverse-transcriptase PCR (qRT-

PCR) were performed according to our previously described method

(Cheng et al.,

; Du et al.,

; Li et al.,

). Mice were killed

by cervical dislocation. Total RNA was extracted from the pancreatic

islets and primary islet

cells derived from

Sst-Cre

fl/fl

, Sst-

Cre

fl/fl

, Arrb1

Arrb2

and wild-type mice by using TRIzol

reagent (Invitrogen, Carlsbad, USA). cDNA underwent reversed tran-

scription with the qRT-PCR Kit (Toyobo, FSQ-101, Osaka, Japan).

qRT-PCR was performed by FastStart SYBR Green Master (Roche,

Basel, Switzerland) on a LightCycler qPCR system (Bio-Rad, Hercules,

USA) to examine the mRNA expression of G proteins (

Gs, Gq, G

, Go,

Gi1, Gi2, Gi3, G12, G13, G15, Gt1, Gt2

and

);

-arrestins (

-arrestin1

and

-arrestin2)

; transcription factors (

Arnt, Foxa2, Foxo1, Foxo3, Klf4,

Nfatc2, Stat6, Tcfl2

and

Tcf3

); synaptotagmin protein subtypes (

Syt1,

Syt2, Syt4, Syt5, Syt7, Syt9, Syt11, Syt13, Syt14, Syt15, Syt16

and

Syt17

);

cell markers (

Nkx6.1, Pdx1, Foxo1, Glut2, Chrm3, Glp1r, Gck,

Kcnj11

and

Adcy8

); proliferation and cell cycle markers (

Ccnd1, Ccnd2,

Cdk2

and

Mki67

);

Crhr2

;

Insulin

Sst

. The relative mRNA level of a

target gene was calculated using 2

ΔΔ

method. All data were from

three independent experiments.

2.4

Immunofluorescence staining and confocal

microscopy

Mice were killed by cervical dislocation. The method of islet isolation

was as described previously (Cheng et al.,

). Pancreatic islets iso-

lated

from

wild-type

mice

infected

with

lentivirus

encoding

mir30-shRNA (G

)

fl/fl

-GFP (G

-shRNA-GFP

fl/fl

) or control virus

WANG

ET AL

(control-shRNA-GFP

fl/fl

) were stimulated with 10-nM UCN3, 100-nM

UCN3 or vehicle for 72 h. The immunofluorescence staining or confo-

cal microscopy were carried out using an LSM880 laser scanning con-

focal microscope system (ZEISS, Germany) according to our previous

descriptions (Cheng et al.,

; Du et al.,

; Li et al.,

). Pan-

creatic islet sections were incubated in 4

C and blocking buffers for

90 min and incubated overnight at 4

C with Somatostatin Polyclonal

Antibody (Thermo Fisher Scientific Cat# PA5-97200,

RRID:AB_

2809002

, 1:200, Waltham, USA), GFP (B-2) (Santa Cruz Biotechnol-

ogy Cat # sc-9996,

RRID:AB_627695

, 1:200, Dallas, USA), or Insulin

Monoclonal Antibody (ICBTACLS), Alexa Fluor

™

488, eBioscience

(Thermo Fisher Scientific Cat# 53-9769-82 [also 53-9769],

RRID:AB_

2574469

, 1:200), anti-Ki67 antibody (Abcam Cat# ab15580,

RRID:

AB_443209

, 1:200, Cambridge, UK) primary antibodies. After rinsing

with PBS for 3 times, the corresponding secondary antibodies

(Donkey anti-Mouse IgG (H

L) Highly Cross-Adsorbed Secondary

Antibody, Alexa Fluor

™

568, Thermo Fisher Scientific Cat# A10037,

RRID:AB_11180865

, 1:200; Donkey anti-Mouse IgG (H

L) Highly

Cross-Adsorbed Secondary Antibody, Alexa Fluor

™

488, Thermo

Fisher Scientific Cat# A-21202,

RRID:AB_141607

, 1:200; Donkey

anti-Rabbit IgG (H

L) Highly Cross-Adsorbed Secondary Antibody,

Alexa Fluor

™

568, Thermo Fisher Scientific Cat#A10042,

RRID:AB_

2534017

, 1:200; Donkey anti-Rabbit IgG (H

L) Highly Cross-

Adsorbed Secondary Antibody, Alexa Fluor

™

488, Thermo Fisher Sci-

entific Cat# A-21206 (also A21206),

RRID:AB_2535792

, 1:200) were

incubated with pancreatic islet sections at room temperature for 2 h

after washing with PBS 3 times, and sections were stained with 4

, 6

diamino-2-phenylindoles.

To study the colocalization of the CRF

receptor and

-arrestin1

-arrestin1 and SYT1, pancreatic TGP52 cells (islet

cell lines,

ATCC Cat# CRL-1573,

RRID:CVCL_0045

) overexpressing CRF

receptor-YFP and

-arrestin1-RFP or SYT1-YFP and

-arrestin1-RFP

were cultured in 35 mm fibronectin coated glass bottom dishes at a

density of 5 x 10

cells per disk 24 h after transfection. After another

24 h,

TGP52

cells

overexpressing

CRF

receptor-YFP

and

-arrestin1-RFP or SYT1-YFP and

-arrestin1-RFP were treated with

UCN3 for 0, 5 and 20 min, respectively. Then, the cells were fixed,

stained with 4

, 6

-diamino-2-phenylindole and analysed by fluores-

cence microscopy.

The Immuno-related procedures used comply with the recom-

mendations made by the

British Journal of Pharmacology

(Alexander

et al.,

2.5

In vitro mouse islet culture

Mouse

pancreatic

islets

were

isolated

from

WT,

Sst-Cre

fl/fl

, Sst-Cre

fl/fl

, Arrb1

Arrb2

mice, STZ mice

(generated as described above) or non-obese diabetic (NOD) mice and

cultured as previously described (Cheng et al.,

; Du et al.,

Lin et al.,

). The islets were collected with the help of a stereo-

scopic microscope and cultured in DMEM medium supplemented with

5.56-mM glucose (Biological Industries, Kibbutz Beit Haemek, Israel),

10% FBS (Gibco, New York, USA) and 0.1% penicillin/streptomycin

(Biological Industries).

2.6

Pancreatic islet

cells isolation

For the quantification of primary pancreatic islet

cells, we isolated

islets from

Sst-Cre

Rosa

lsl-RFP

mice or

Sst-Cre

Rosa

lsl-RFP

mice

pretreated with streptozocin, then dispersed into single cell suspen-

sions

incubation

with

0.6-U

Dispase

(Roche

04942078001)

according

our

description

(Cheng

et al.,

), then the islet single cells were washed and flow cyto-

metric analyses were performed on a cytoflex (Beckman Coulter, Brea,

USA). To isolate the primary islet

cells, we use the

Sst-Cre

Rosa

lsl-

RFP

mice or

Sst-Cre

Rosa

lsl-RFP

mice pretreated with streptozotocin,

the RFP

population was isolated from the islet signal cell suspension

by flow cytometry (Beckman Coulter). The isolated RFP

cells were

subjected to qRT-PCR analysis.

2.7

Constructs of CRF2 receptor and RyR3

Mouse

Crhr2

Arrb1

and G protein subunits (G

s-RLuc8, G

q-RLuc8,

11-RLuc8, G

3 and G

9-GFP2) genes were subcloned into the

pcDNA3.1 expression vectors. CRF

receptor-YFP,

-arrestin1-RFP,

SYT1-YFP, CRF

receptor-G

11 and CRF

receptor-G

q were created

by fusion or insertion of different sequences to constructs according

to our previous descriptions (Xiao et al.,

; Xiao et al.,

). The

segment Ryr3-2616-2820, the segment Ryr3-1-543 and the segment

Ryr3-4592-4868 were cloned into the pcDNA3.1 expression vectors

for phosphorylation examination. All of the constructs and mutations

were verified by DNA sequencing.

2.8

Lentivirus infection

The isolated pancreatic islets from

Sst-Cre

mice were administered

with G11-shRNA-GFP

fl/fl

lentivirus (OBiO) for at least 48 h followed

by continued culture for 3

–

5 days until fluorescence could be

observed under a fluorescence inverted microscope (ZEISS) to ensure

sufficient infection according to our previous described methods (Du

et al.,

). After infection, islets were prepared for further experi-

ments including SST secretion assay, insulin secretion assay, and qRT-

PCR.

2.9

Glucose-stimulated insulin secretion

The Glucose-stimulated Insulin Secretion (GSIS) methodology is simi-

lar to the description we have published before (Cheng et al.,

;

Du et al.,

; Lin et al.,

). The cultured mice islets were prein-

cubated with 1- or 20-mM glucose, then treated with 10 nM or

100 nM UCN3.

Sst-Cre

mouse islets were infected with lentivirus

WANG

ET AL

encoding G

-shRNA-GFP

fl/fl

or control lentivirus, isolated then pre-

treated with different concentrations of UCN3 or control vehicle for

72 h before GSIS measurement. The supernatant was collected

30 min after stimulation. Insulin levels were measured using the

rat/mouse insulin enzyme-linked immunosorbent Assay kit (Millipore,

EZRMI-13 K) according to the method provided by the manufacturer.

Insulin secretion data were normalized to insulin content or expressed

as an insulin stimulation index.

2.10

Somatostatin (SST) secretion

Somatostatin secretion was measured according to previously

reported methods (Du et al.,

; Li et al.,

; Lin et al.,

Pancreatic islets were isolated from wild-type (WT), Sst-Cre

fl/fl

Sst-Cre

fl/fl

, Arrb1

or Arrb2

mice or islets isolated from

wild-type mice infected with G11-shRNA-GFP

fl/fl

or control virus for

at least 48 h cultured in low glucose DMEM media. Diameters of the

islets varied: approximately 80% of islets were between 50 and 150

m, approximately 20% of islets were between 150 and 250

m, and

very few islets had diameters less than 50

m or more than 250

Moreover, results indicated that the secretion lever of SST induced by

UCN3 was proportional to the diameter of the islet, and the larger the

diameter of the islet, the higher the level of SST secretion induced by

UCN3 in a more accurate manner, we examined the SST secretion

with islets (diameter: 50

–

150

m) isolated from WT, Sst-Cre

fl/fl

Sst-Cre

fl/fl

, Arrb1

or Arrb2

mice in response to UCN3

stimulation. Islets were stimulated with UCN3(0, 1, 10 or 100 nM),

pretreated with Astressin 2B (A2B) (100 nM). Rp-cAMPS (50

M),

H89 (500 nM), following the manufacturer's instruction. For SST con-

tent measurement, the islets were lysed with 50

l cold acid ethanol

solution as previously described (Cheng et al.,

; Du et al.,

Li et al.,

). After overnight incubation at 4

C, the supernatants

were collected by 12,000 rpm centrifugation and were neutralized by

1 M Tris (pH 7.5). The SST levels were measured with the ELISA kits

(Phoenix Pharmaceuticals, #EK-060-03, Burlingame, USA) following

the manufacturer's instructions.

2.11

RNA interference (RNAi)

TGP52 cells were transfected with siRNAs to knock down targeted

genes (including

Gnas, Gnaq, Gna11, Arrb1, Arrb2, Ryr1, Ryr2, Ryr3,

Stat6, Foxo3, Tcf3, Syt1, Syt2, Syt7, Syt9, Syt13, Syt14, Syt15

and

Syt16

). In order to achieve a better knock down effect, we first

compare three designed siRNAs, picking out the one with the

best efficiency in cell line knock-down. Then, we performed the

siRNA transfection twice, with the second RNAi transfection 24 h

after the first transfection. This optimized protocol enabled knocking

down targeted genes (including

Gnas, Gnaq, Gna11, Arrb1, Arrb2,

Ryr1, Ryr2, Ryr3, Stat6, Foxo3, Tcf3, Syt1, Syt2, Syt7, Syt9, Syt13,

Syt14, Syt15

and

Syt16

) achieves at relatively high efficiency com-

pared with siRNA transfection only once. After transfection, cells

were prepared for SST secretion detection in response to different

ligands or drugs.

2.12

-G

dissociation assay

BRET assays were performed using a G-protein dissociation system as

previously described by our laboratory (Cheng et al.,

; Mao

et al.,

; Yang et al.,

). G

-RLuc8, G

3, and G

9-GFP2)

and G

-RLuc8, G

3, G

9-GFP2) BRET assays were also per-

formed as previously described (Mao et al.,

). The experimental

plasmids were co-transfected into HEK293 cells (ATCC Cat# CRL-

1573,

RRID:CVCL_0045

, Rockefeller, USA) with the CRF

receptor,

-RLuc8, G

3, and G

9-GFP2. Forty-eight hours after transfection,

the cells were digested with 0.25% trypsin, washed three times, stimu-

lated with UCN3 at the indicated concentrations (10

–

M). The

BRET signal was measured after the addition of the luciferase sub-

strate coelenterazine 400a (5

M) using a Mithras LB940 microplate

reader (Berthold Technologies, Vilvoorde, Belgium) equipped with

BRET filter sets and calculated as the ratio of light emission at

510/400 nm according to our previous description (Xiao et al.,

;

Yang et al.,

2.13

-arrestin1 recruitment assay

BRET assays were performed using the

-arrestin1 recruitment sys-

tem previously described by our laboratory (Cheng et al.,

). The

plasmid CRF

receptor-YFP and

-arrestin1-Rluc or CRF

receptor,

-arrestin1-Rluc and Synaptotagmin1-YFP were co-transfected into

HEK293 cells. Twenty-four hours after transfection, the medium was

changed again to DMEM complete medium and incubated for 24 h at

C in a 5% CO

humidified cell incubator. Different concentrations

of UCN3 were configured using PBS buffer. The medium was dis-

carded, washed once with PBS buffer, and the cells were digested

with 0.25% trypsin and terminated with DMEM complete medium.

The cells were centrifuged at 1000 rpm for 3 min at room tempera-

ture, the supernatant was discarded and the cells were resuspended

using PBS, and the cells were washed twice by further centrifugation.

The cells resuspended in 80-

L PBS were added to the 96-well cell

culture plate, and then 10-

L ligand was added to the incubator at

C for 10 min. After the reaction was terminated on ice, 10-

L sub-

strate was added. Signals from excitation light at 480 nm and emission

light at 530 nm were immediately detected using a MithrasLB940

microplate

reader

(BertholdTechnologies).

The

BRET

signal

was calculated as the ratio of light emission at 530/480 nm (Yang

et al.,

2.14

Glosensor-cAMP assay

Synergy measurements were performed using the GloSensor system,

previously described by our laboratory and others (Cheng et al.,

WANG

ET AL

GloSensor and CRF

receptor plasmids were co-transfected into

HEK293 cells at a ratio of 1: 2.5. Twenty-four hours after transfection,

transfected HEK293 cells were seeded into 96-well cell culture plates

and incubated for 24 h. The transfected cells were pre-incubated with

L of DMEM medium containing GloSensor cAMP reagent

(Promega) at 37

C for 2 h. UCN3 at different concentrations as speci-

fied in the text or figure legends was configured using HEPES buffer.

Luminescence per well per second (LCPS) was read on a multimode

microplate detector (PerkinElmer EnVision). The data were analysed

using the Sigmoid dose

–

response function in GraphPadPrism7.0.

2.15

Co-immunoprecipitation

TGP52 cells after starvation were stimulated with 100 nM UCN3 for

different times (0, 2, 5, 10 and 30 min) and prepared for detecting of

RyR3 or pPKA. HEK293 cells after starvation were stimulated with

100 nM UCN3 to the CRF

receptor,

-arrestin1 or SYT1. TGP52 cells

were lysed with Lysis buffer (with protease inhibitors) on ice for

30 min. The supernatants were prepared by centrifugation. The 2

anti-ryanodine receptor3 Antibody (

RyR3

) (Alomone Labs Cat# ARR-

003,

RRID:AB_2040186

) was added to each group, and the mixture

was vertical mixed at 4

C for 6 h. Then 20-

L ProteinA/G beads was

vertical mixed at 4

C for 2

–

4 h. Beads and binding proteins were

washed with cold buffer to exclude interference from non-specific

binding proteins. After adding loading buffer, the specimens were

denatured at 37

C for 30 min and subsequently subjected to western

blot analysis using specific antibodies against RyR3 and pPKA

(Phospho-(Ser/Thr) PKA Substrate Antibody, Cell Signaling Technol-

ogy Cat# 9621 (also 9621S, 9621L),

RRID:AB_330304

HEK293 cells were transiently transfected with plasmids encod-

ing HA-

-arrestin1 and the CRF

receptor or plasmids encoding MYC-

SYT1. After 48 h, the cells were stimulated with 100 nM UCN3 or

vehicle for 10 min, use same methods previously described by our lab-

oratory to deal (Cheng et al.,

) and subsequently subjected to

western blot analysis using specific antibodies against the CRF

recep-

tor Polyclonal Antibody (Thermo Fisher Scientific Cat# PA5-23129,

RRID:AB_2540656

-arrestin1 Monoclonal Antibody (

-arrestin1)

(Thermo Fisher Scientific Cat# 39-5000

RRID:AB_2533416

) and

Synaptotagmin-1 (D33B7) Rabbit mAb (MYC-SYT1) (Cell Signaling

Technology Cat# 14558,

RRID:AB_2798510

2.16

Western blotting

Western blotting were performed according to previously described

methods (Cheng et al.,

; Du et al.,

; Li et al.,

). We

examined G

protein levels in TGP52 cells transfected with sicontrol,

siFoxo3, siStat6 and siTcf3, 48 h after transfection, the cells were

washed twice with cold PBS, then lysated the cells and use antibodies

against G

protein (G alpha 11 Antibody [D-6], Santa Cruz Biotech-

nology Cat# sc-390382,

RRID:AB_2715558

) to characterize the

respective G11 protein levels.

For examination of phosphorylated protein level detected by

PKA-specific substrate phosphorylation antibody, the plasmid encod-

ing the CRF

receptor was co-transfected into HEK293 cells with the

RyR3 fragments, and experiments were performed 48 h after trans-

fection. Cells were starved using serum-free DMEM medium for 8 h

and then starved with PBS buffer for 1

–

2 h, then PKA-specific sub-

strate phosphorylation antibody was used for examination.

The TGP52 cells were stimulated with 100-nM UCN3 for differ-

ent times (0, 2, 5, 10 and 30 min) and terminated on ice. The cells

were lysed with lysis buffer (with protease inhibitors) on ice for

30 min, then centrifuged to remove the supernatant. The 15-

L Flag

Beads was added and mixed vertically at 4

C for 6 h. The Beads and

binding proteins were washed with cold buffer to exclude interfer-

ence from non-specific binding proteins. After adding loading buffer,

the cells were denatured at 100

C for 10 min and subsequently sub-

jected to western blot.

2.17

Chromatin immunoprecipitation (ChIP)

ChIP experiments were performed using methods previously described

(Li et al.,

). Cultured TGP52 cells were aspirated to discard the

medium and washed three times with PBS buffer. Cross-linking was

performed with 1% formaldehyde for 15 min at room temperature.

Cells were washed three times with cold PBS buffer and scraped, and

then centrifuged (12,000 rpm, 4

C, 20 min) to remove cell precipitates.

The cells were sonicated and centrifuged (12,000 rpm, 4

C, 20 min) and

the supernatant was retained and then incubated with 2

g STAT6 anti-

body overnight. Protein A/G Beads were added and mixed vertically at

C for 2

–

4 h. The complexes were washed with low and high salt

buffers. To profile the binding patterns of transcription factor STAT6 in

the gene region of G

, we design different primers based on the DNA

region 1999-1714, 1737-1476, 1476-1214, 1219-843, 849-633,

654-345, 365-11 downstream of the transcription initiation sites of the

and DNA was extracted and precipitated for subsequent RT-PCR

and other assays.

2.18

BRET-based ligand binding assay

BRET-based ligand binding assay for the N-terminal NanoLuc (NLuc)-

tagged CRF

receptor and C-terminal FITC-conjugated UCN3 was

performed as previously described (Cheng et al.,

; Strachan

et al.,

; Yang et al.,

). Briefly, HEK293 cells were transiently

transfected with NLuc-CRF

receptor. For saturation binding, the cells

were treated with increasing concentrations of UCN3

–

FITC in the

presence or absence of 3

M unlabeled UCN3. For competitive bind-

ing, the cells were transfected with CRF

receptor-G

11 or CRF

receptor-G

q and treated with increasing concentrations of UCN3

–

FITC. Luciferase substrate coelenterazine-h (5 mM) was added before

light emissions were recorded using a Mithras LB940 microplate

reader (Berthold Technologies). The BRET signal was determined by

calculating the ratio of luminescence at 530 nm/460 nm. After fitting

WANG

ET AL

the competition binding curves, the Ki of the UCN3 on the CRF

receptor or CRF

receptor-G

complex were calculated by using the

Cheng-Prusoff equation:

IC50

where [L] is the UCN3-FITC concentration (nanomolar), IC50 is the

median inhibitory concentration, and Kd is the dissociation constant

of UCN3-FITC generated from the saturation binding experiment. K

indicated the binding of the ligand to the receptor alone, K

resem-

bles the binding of the UCN3 to the CRF

receptor-Gq or CRF

receptor-G

complexes.

2.19

Data and statistical analysis

Data are presented as mean ± SEM, and two or more groups were

compared by Student's

-test or one-way ANOVA by using GraphPad

Prism. In all animal experiments, statistical analyses were conducted

on datasets with a minimum group size of n

≥

5, while in all cell exper-

iments, statistical analyses were performed on datasets with a mini-

mum group size of n

≥

6. A significance level of

< 0.05 was

considered statistically significant for all analyses. Post-hoc tests were

run only if F achieved P<0.05 and there was no significant variance

inhomogeneity. Data and statistical analyses were conducted in accor-

dance with the recommendations of the

British Journal of Pharmacol-

ogy

on experimental design and analysis in pharmacology (Curtis

et al.,

http://www.guidetopharmacology.org

). All western blotting experiments conform to the guide-

lines of the

British Journal of Pharmacology

2.20

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corre-

sponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY

and are permanently archived

in the Concise Guide to PHARMACOLOGY 2023/23 (Alexander,

Christopoulos et al.,

; Alexander, Fabbro et al.,

; Alexander,

Kelly et al.,

; Alexander, Mathie et al.,

R E S U L T S

3.1

Specific G protein and Arrestin subtypes

contribute to the secretion of somatostatin (SST)
induced by 100 nM UCN3

Accompanied by insulin, UCN3 is synthesized by

cells within pan-

creatic islets, packaged in vesicles and released in response to high

glucose or other physiological/pathological stimulation to promote

somatostatin secretion from

cells (Li et al.,

2007

;Li et al.,

; van

der Meulen et al.,

). It has been reported that the secretion of

UCN3 from cultured

cells is stimulated by high glucose, forskolin

and insulin secretagogues, such as GLP-1, carbachol (a cholinergic

agonist) and KCL. Previous study indicated that the UCN3 concentra-

tion in wild-type mouse plasma is approximately 2.5

–

5 nM (Flaherty

et al.,

). We then measured the concentration of UCN3 in mouse

plasma by Elisa assay, and the concentration is determined as 2.07

± 0.09 nM (Figure

S2a

). Importantly, UCN3 secreted by pancreatic

cells could signal to islet

cells in a paracrine manner through the acti-

vation of CRF

receptors, a class B GPCR (Li et al.,

; van der

Meulen et al.,

). Engagement of the CRF

receptor by UCN3 has

been reported to activate multiple G protein and arrestin subtypes

(Zhao et al.,

), which include but are not limited to Gs, Gq,

-arrestin1 or

-arrestin2, to elicit distinct downstream functions. To

understand how the activation of CRF

receptors leads to intracellular

signal changes and functional outcomes of somatostatin-producing

pancreatic

cells, we profiled the expression levels of different G pro-

tein and arrestin subtypes in

Sst-Cre

Rosa

lsl-RFP

-labelled pancreatic

islet somatostatin-producing pancreatic

cells. The results indicated

that

Arrb1

Arrb2

and

have similar expres-

sion levels in

cells and brain tissue and that

Sst

and

Crhr2

are more highly expressed in

cells than in brain tissue. In contrast,

the other three tested G protein subtypes had lower expression levels

cells than in brain tissue. We used brain tissue as a control

because brain expressed abundant GPCRs and G proteins (Zhang

et al.,

) (Figure

S2b,c

We then investigated the expression changes in these G protein

or arrestin subtypes in response to physiological stimulation and

under different pathological conditions using mouse models. In

response to 100 nM UCN3 (an excessive concentration of UCN3

in our study) stimulation, the mRNA levels of

Arrb1

and

Arrb2

Sst-Cre

Rosa

lsl-RFP

labelled pancre-

atic

cells increased (Figure

). In contrast, the mRNA levels of

Arrb1

and

Arrb2

Sst-Cre

Rosa

lsl-RFP

labelled cells

decreased in diabetic models of NOD mice or multiple low-dose

streptozocin (MLD-STZ)-treated mice. We also found that STZ mice

and NOD mice showed poor response to UCN3 stimulation of SST

secretion compared with that in WT mice (Figure

S2d,e

), which may

be related to the decreased mRNA level of

Gs, Gq/

and

Arrb1/2

STZ mice or NOD mice compared with WT mice (Figure

1b,c

). These

results suggested that the G protein/arrestin-biased signalling system

was greatly disturbed by both long-term UCN3 treatment and in dia-

betic models (Figure

We next investigated how different G protein- or arrestin-

mediated CRF

receptor signalling pathways contributed to SST

secretion in response to 100 nM UCN3 stimulation. Importantly, the

diameter of the islets varies: approximately 80% islets were between

–

150

m, approximately 20% islets were between 150 and

250

m, and very few islets had diameters less than 50

m or more

than 250

m (Figure

S2f

). Moreover, our data indicated that the

secretion level of SST induced by UCN3 was proportional to the diam-

eter of the islet, and the larger the diameter of the islet, the higher the

level of SST secreted by the islet stimulated by UCN3 (Figure

S2g

Importantly, 100-nM Astressin 2B (A2B), a specific antagonist of the

WANG

ET AL

S2h,i

). Moreover, whereas knocking out

-arrestin2 in

Arrb2

mice

had no significant effects,

Arrb1

deficiency in pancreatic islets from

Arrb1

mice significantly reduced 100 nM UCN3-induced SST

secretion (Figures

1i,j

and

S2j

). These results suggested that Gs-, G

and

-arrestin1-mediated

-cell signalling contributed to 100-nM

UCN3-stimulated SST secretion in primary pancreatic islets.

We next knocked down selective G protein subtypes or arrestins

via siRNAs and pharmacologically blocked Gs signalling by NF449, a

specific Gs inhibitor, to investigate the contribution of 100-nM

UCN3-stimulated SST secretion in TGP52 cells. Despite glucose

induced SST secretion in TGP52 cells, these are not comparable to

primary pancreatic

cells, but we speculated that studies in TGP52

cell could be a good supplement to primary islet studies. At least, high

glucose induced SST secretion in TGP52 cells was significantly

increased in response to UCN3 compared with these treated with

vehicle and no better pancreatic

cell lines were commercially avail-

able (Figure

S2k

). The results repeated those observed in isolated

pancreatic islets, which suggested that Gs, G

and

-arrestin1 signal-

ling contributed to 100 nM UCN3-induced SST secretion in the

TGP52 cell lines (Figures

and

S2k

3.2

Differential mechanisms underlying

somatostatin (SST) secretion in response to 10-nM
UCN3 stimulation

Because reported and our measured plasma level of UCN3 ranged

from 2

–

5 nM in wild-type mouse plasma (Figure

S2a

), we measured

SST secretion in response to UCN3 concentrations of 2 and 10 nM.

Notably,

both

and

10-nM

UCN3

were

able

elicit

UCN3 secretion in isolated islets, which was blocked by the applica-

tion of 100-nM Astressin 2B (A2B) (Figure

). This result suggested

that a low concentration of UCN3 was able to elicit SST secretion via

CRF

receptors in pancreatic islets.

F I G U R E 1

Specific G protein or arrestin subtypes contribute to the secretion of somatostatin (SST) induced by UCN3. (a) The relative

expression levels of G proteins and arrestins in islet

cells in response to UCN3 stimulation were detected by qRT-PCR. Islet

cells were labelled

with RFP in

Sst-Cre

Rosa

lsl-RFP

mice. Approximately 2 * 10

RFP

cells isolated from 6

–

Sst-Cre

Rosa

lsl-RFP

mice (1 * 10

RFP

cells per

group) by flow cytometry, stimulated with UCN3 (100 nM) for 72 h and then prepared for qRT-PCR examination. The data are from three
independent experiments (n

3). (b,c) The relative expression levels of G proteins and arrestins in islet

cells from STZ (b) or NOD (c) mice were

detected by qRT-PCR. Islet

cells were labelled with RFP in

Sst-Cre

Rosa

lsl-RFP

mice. STZ mice were obtained from

Sst-Cre

Rosa

lsl-RFP

mice

treated with STZ (20 mg

day

) for 5 days using

Sst-Cre

Rosa

lsl-RFP

mice treated with control vehicle as a control. Non-obese diabetic

(NOD) mice were crossed with

Sst-Cre

Rosa

lsl-RFP

mice to label islet

cells with RFP. Approximately 1 * 10

RFP

cells isolated from 6

–

Sst-

Cre

Rosa

lsl-RFP

mice treated with STZ (20 mg

day

) for 5 days or 6

–

8 non-obese diabetic (NOD) mice were crossed with

Sst-Cre

Rosa

lsl-

RFP

mice or 3

–

Sst-Cre

Rosa

lsl-RFP

mice by flow cytometry and prepared for qRT-PCR detection. The data are from three independent

experiments (n

3). (d,e) Somatostatin secretion in response to UCN3 or astressin 2B (A2B, CRF

receptor receptor antagonist, 100 nM)

stimulation were detected in islets isolated from

Sst-Cre

fl/fl

mice (d),

Sst-Cre

fl/fl

mice (e). Islets diameters with radius ranging from 50

–

150

m were selected. WT or

Sst-Cre

mouse islets infected with lentivirus encoding non-sense shRNA were prepared as control. Three

hundred islets from 6

–

8 mice were grouped from WT,

Sst-Cre

fl/fl

Sst-Cre

fl/fl

Sst-Cre

control-shRNA-GFP

fl/fl

and

Sst-Cre

G11-shRNA-GFP

fl/fl

mice (100 islets per group). The data are from six independent experiments (n

6). (f) Immunostaining of GFP (green) cells

and SST (red) cells in islets isolated from

Sst-Cre

mice were infected with G11-shRNA-GFP

fl/fl

and control-shRNA-GFP

fl/fl

lentivirus for 48 h

and cultured for another 3

–

5 days. The right panel is Pearson's correlation analysis of G

-shRNA-GFP

fl/fl

and SST fluorescence intensities. The

Pearson's correlation coefficient was 0.71. Scale bar: 50

m. The 100 islets isolated from 2

–

3 mice were used for slicing and counted as an

independent experiment; 4

–

7 random areas were selected from each section and 10 sections were randomly selected from each mouse. The data

are from six independent experiments (n

6). (g) Quantification of GFP cells and SST cells in islets isolated from

Sst-Cre

mice were infected

with G11-shRNA-GFP

fl/fl

and control-shRNA-GFP

fl/fl

lentivirus for 48 h and cultured for another 3

–

5 days. The 100 islets isolated from 2

–

3 mice

were used for slicing and counted as an independent experiment; 4

–

7 random areas were selected from each section and 10 sections were

randomly selected from each mouse. The data are from six independent experiments (n

6). (h) Somatostatin secretion in response to UCN3 or

astressin 2B (A2B, CRF2 receptor receptor antagonist, 100 nM) stimulation were detected in islets isolated from

Sst-Cre

mice islets infected

with lentivirus encoding G

-shRNA-GFP

fl/fl

. WT or

Sst-Cre

mice islets infected with lentivirus encoding non-sense shRNA were prepared as

control. Islets diameters with radius ranging from 50

–

150

m were selected. Three hundred islets from 6

–

8 mice were grouped from

Sst-Cre

control-shRNA-GFP

fl/fl

and

Sst-Cre

-shRNA-GFP

fl/fl

mice (100 islets per group). The data are from six independent experiments (n

6).

(i) SST secretion from islets isolated from WT mice or

Arrb1

mice in response to stimulation with UCN3 or A2B. Islets diameters with radius

ranging from 50

–

150

m were selected. The 150 islets from 2

–

Arrb1

mice or WT mice treated with UCN3 or A2B were grouped (50 islets

per group). The data are from six independent experiments (n

6). (j) SST secretion from islets isolated from WT mice or

Arrb2

mice in

response to stimulation with UCN3 or A2B. Islets diameters with radius ranging from 50

–

150

m were selected. The 150 islets from 2

–

Arrb2

mice treated with UCN3 or A2B were grouped (50 islets per group). The data are from six independent experiments (n

6).

(k) Somatostatin secretion in response to UCN3 or A2B stimulation were detected in TGP52 cells with G proteins or Arrestins knockdown.
TGP52 cells were transfected with siRNAs targeted to

Gnas

Gnaq

Gna11

Arrb1

Arrb2

for 48 h. Then cells were treated with UCN3 (100 nM),

A2B (100 nM) or NF449 (1

M) for 1 h. The data are from three independent experiments (n

3). (d,e) *

< 0.05, n.s. no significant difference.

Relative somatostation levels from

Sst-Cre

fl/fl

mice (d),

Sst-Cre

fl/fl

mice (e) compared with WT mice. (h) *

< 0.05, n.s. no significant

difference.

Sst-Cre

control-shRNA-GFP

fl/fl

mice compared with

Sst-Cre

-shRNA-GFP

fl/fl

mice. (i,j) *

< 0.05, ns, no significant difference;

Arrb1

mice (i) or

Arrb2

mice (j) compared with WT mice. The bars indicate the mean ± SEM values. The data were analysed to determine

significance via two-way ANOVA with Dunnett's post hoc test. Statistical tests were not carried out where n was less than 5.

WANG

ET AL

F I G U R E 2

Effects of UCN3 at

‘

low

’

concentration on somatostatin (SST) secretion. (a) Somatostatin secretion of mice islets in response to

different concentrations of UCN3 treated with or without the CRF

receptor antagonist Astressin 2B (A2B). Islet diameters with radius ranging from

–

150

m were selected. Three hundred islets from 6

–

8 mice were grouped from WT mice (100 islets per group). Islets were isolated and stimulated

with UCN3 (0, 2, 10 nM) or A2B (100 nM) for 1 h. The data are from six independent experiments (n

6). (b) SST secretion from islets isolated from

Sst-Cre

mouse islets infected with lentivirus encoding G

-shRNA-GFP

fl/fl

or infected with lentivirus encoding non-sense shRNA in response to

stimulation with 10-nM UCN3. Islet diameters with radius ranging from 50

–

150

m were selected. The 100 islets from 2

–

Sst-Cre

mice infected

with lentivirus encoding G

-shRNA-GFP

fl/fl

or infected with lentivirus encoding non-sense shRNA (50 islets per group). The data are from six

independent experiments (n

6). (c) SST secretion from islets isolated from

Sst-Cre

fl/fl

mice or

Sst-Cre

mice in response to stimulation with

10-nM UCN3. Islets diameters with radius ranging from 50

–

150

m were selected. The 100 islets from 2

–

Sst-Cre

fl/fl

mice or

Sst-Cre

mice

treated with UCN3 were grouped (50 islets per group). The data are from six independent experiments (n

6). (d) SST secretion from islets isolated

from WT mice or

Arrb1

mice in response to stimulation with 10 nM UCN3. Islet diameters with radius ranging from 50

–

150

m were selected.

100 islets from 2

–

Arrb1

mice or WT mice treated with UCN3 were grouped (50 islets per group). The data are from six independent experiments

6). (e) Somatostatin secretion in response to UCN3 or Rp-cAMPS stimulation were detected in TGP52 cells with

Arrb1

Gna11

knocking down.

TGP52 cells were transfected with siRNAs targeted to

Arrb1

Gna11

for 48 h. Then cells were treated with UCN3 (10 nM) or Rp-cAMPS (50

cAMP inhibitor) for 1 h. The data are from three independent experiments (n

3). (a) *

< 0.05, n.s. no significant difference. Islet treated with A2B

compared with control (Con). (b) n.s. no significant difference. Islets isolated from

Sst-Cre

mice infected with lentivirus encoding G

-shRNA-GFP

fl/fl

compared with infected with lentivirus encoding non-sense shRNA. (c) *

< 0.05, n.s. no significant difference.

Sst-Cre

fl/fl

mice compared with

Sst-

Cre

mice. (d) *

< 0.05, n.s. no significant difference.

Arrb1

mice compared with WT mice. The bars indicate the mean ± SEM values. All data

were analysed by two-sided one-way ANOVA with Tukey's test. Statistical tests were not carried out where n was less than 5.

WANG

ET AL

In contrast to the contribution of G

signalling to 100-nM

UCN3-induced SST secretion, G

signalling showed no significant

effects on 10-nM UCN3-induced SST secretion, because treatment

with 10 nM UCN3 did not significantly alter SST secretion in islets of

Sst-Cre

-shRNA-GFP

fl/fl

virus compared with the islets of

Sst-

Cre

control-shRNA-GFP

fl/fl

virus (islets of

Sst-Cre

mice were

infected with G

-shRNA-GFP

fl/fl

virus or control-shRNA-GFP

fl/fl

virus, respectively) (Figure

). However, SST secretion induced by

10-nM UCN3 was significantly decreased in primary islets derived

from

Sst-Cre

fl/fl

mice or

Arrb1

mice and was significantly

lower than that in their wild-type littermates (Figure

2c,d

). Moreover,

the application of either Rp-cAMPS or an siRNA targeting

Arrb1

but

not G

decreased SST secretion in TGP52 cells (Figure

). Rp-

cAMPS is inhibitor of secondary messenger cAMP downstream signal-

ling. Therefore, these results suggested that Gs and

-arrestin1-

signalling but not G

signalling contributed to SST secretion in islets

in response to 10-nM UCN3 stimulation.

3.3

The Gs-PKA-RyR3 pathway contributes to

UCN3-induced somatostatin (SST) secretion

To investigate the molecular mechanisms underlying Gs signalling-

mediated SST secretion in response to UCN3 stimulation, we used

the GloSensor assay to examine the cellular cAMP level in response

to different treatment times and different UCN3 concentrations. In

CRF

receptor-overexpressing HEK293 cells, the intracellular cAMP

level peaked between 6 and 10 min and then diminished at approxi-

mately 20 min in response to UCN3 stimulation at different concen-

trations. The EC

value of UCN3-elicited intracellular cAMP

accumulation that was derived from the concentration-dependent

curve was 25.5 ± 1.4 nM (Figure

3a,b

). Downstream of cAMP,

PKA

activity was increased, which promoted the phosphorylation of cyclic-

AMP response element-binding protein (

CREB

) to modulate the

expression of a spectrum of genes involved in hormone secretion,

including SST (Kidd et al.,

; Vallejo et al.,

1992

; Wang

et al.,

). We then used CREB phosphorylation at the pS

133

site as

a marker for the activation of the CRF

receptor-Gs-PKA signalling

pathway in response to UCN3. The phosphorylation of CREB showed

an increase beginning at 2 min, peaked between 5 and 30 min, and

then returned to normal at 60 min (Figures

and

S3a

). The EC

value for the UCN3-induced pS

133

level of CREB was 3.434

± 0.08 nM (Figures

and

S3b

). These results indicated that the clas-

sic CRF

receptor-Gs-PKA signalling pathway responded quickly to

low concentrations of UCN3. We next exploited the cAMP inhibitor

Rp-cAMPS and the PKA inhibitor H89 to evaluate how the inhibition

of cAMP or PKA signalling mimicked Gs inhibition by comparing their

effects with those of NF449 and 100 nM UCN3. Importantly, both

Rp-cAMPS and H89 largely reproduced the effects of NF499, sug-

gesting that cAMP-PKA signalling contributed to Gs-mediated SST

secretion downstream of the CRF

receptor (Figure

Previous studies have suggested that the phosphorylation of

ryanodine receptors (RyRs) by PKA participates in insulin secretion.

Importantly, the application of ryanodine or Rp-cAMPS inhibited

UCN3-induced SST secretion, and this effect was abolished in

Sst-

Cre

fl/fl

mice (Figure

). These results strongly suggest that

RyR activity is required for SST secretion downstream of Gs signal-

ling. Among the three RyRs, the mRNA level of

Ryr3

was more

enriched in primary

cells (Figure

S3c

). Moreover, the treatment of

TGP52 cells with RyR3-siRNA decreased UCN3-stimulated SST

secretion, whereas treatment with an siRNA targeting other RYR

subtypes or the control siRNA showed no effects (Figures

and

S3d

–

f).

We then monitored the time course of the phosphorylation of

RyR3 by PKA in HEK293 cells overexpressing CRF

receptors and

treated with 100 nM UCN3. RyR3 was immunoprecipitated, and the

phosphorylation of RyR3 by PKA was examined with an anti-phos-

pho-PKA-substrate antibody. The phosphorylation level of RyR3 by

PKA was predicted to peak at 5 min (an anti-phospho-PKA-substrate

antibody was used) (Figure

). We predicted the phosphorylation

sites of RyR3 by PKA using the software

), which suggested phosphorylation sites related

to S156, S160, S2706, S4697 and T4712. By inspecting the RyR3

structure (solved or predicted by AlphaGo), we selected solvent-

accessible sites for further investigation. According to previous stud-

ies, Ser2809 of human RyR2, Ser2844 of mouse RyR1 and Ser2843 of

human RyR1 are suggested to be the key phosphorylation sites of

PKA. Moreover, sequence alignment of the residues around the con-

stituting phosphate binding site among different RyR family members

showed that the corresponding site on RyR3 was S2706, whereas

S2809 and S2844 of RyR3 were in the conserved sequence. Based on

the predicted phosphorylation sites of RyR3 and reported key sites of

similar RyR1/2 family members, we selected and cloned the segment

RyR3-2616-2820 (encompassing S2706), the segment RyR3-1-543

(encompassing S156, S160), and the segment RyR3-4592-4868

(encompassing S4697, T4712) for further in vitro phosphorylation

examination.

We transfected selective RyR3 fragments that were harbouring

the predicted phosphorylation sites of PKA and examined how their

phosphorylation levels are affected by site-directed mutants. Nota-

bly, the UCN3-induced phosphorylation levels of RyR3 fragments

were specifically diminished by the S156A, S2706A and S4697A

mutations. Moreover, similar to full-length RyR3, the phosphoryla-

tion levels of RyR3-1-543, RyR3-2616-2820 and RyR3-4592-4868

exhibited increases at 5 min in response to UCN3 stimulation

(Figures

and

S3g

). These results indicate that the potential

phosphorylation sites of RyR3 downstream of UCN3-CRF

receptor

signalling include the S156, S2706 and S4697 positions (Figures

and

S3h

). Our results suggested increased phosphorylation of RyR3

by PKA upon UCN3 stimulation. Furthermore, we identified the

S156, S2706 and S4697 sites as potential PKA phosphorylation

sites

downstream

UCN3-CRF

receptor

engagement

(Figure

). Further in vivo or biochemical studies are required to

investigate whether the phosphorylation of these sites by PKA

plays important roles in RyR3 activation and downstream SST

secretion.

WANG

ET AL

3.4

The contribution of STAT6- and

UCN3-biased properties to G

signalling

Our studies in islets suggested that G

-mediated signalling but not

Gq-mediated signalling contributed to UCN3-induced SST secretion.

We recapitulated these phenomena by using siRNAs against Gq and

in TGP52 cells. PLC-IP3-calcium signalling downstream of the Gq

pathway contributes to SST secretion (or insulin secretion from pan-

creatic

cells). Consistent with these observations, we found that

application of the specific PLC inhibitor U73122 showed effects simi-

lar to those of TGP52 cells treated with the G

siRNA and Gq siRNA

or the G

siRNA alone (Figure

). Moreover, UCN3 was able to acti-

vate G

using a G trimer dissociation assay in both a concentration-

and time-dependent manner (Figure

4b,c

). These results suggested

that the G

-PLC pathway contributed to UCN3-induced SST secre-

tion in pancreatic

cells.

Previously, we showed that the direct fusion of arrestin or G pro-

tein subtypes could effectively determine the biased factor of selec-

tive downstream signalling of a particular GPCR, such as the

receptor

(Strachan et al.,

). We therefore probed the bias prop-

erty of UCN3 on Gq and G

signalling after CRF

receptor activation

using ligand binding assays and a chimeric fusion method. We used a

BRET assay to monitor ligand binding to CRF

receptors by fusing

FITC at the UCN3 C-terminus and linking Nluc at the N-terminus of

the CRF

receptor (Figure

S4a,b

) (Cheng et al.,

). Similar to that

previously observed in the AT

receptor (Fu et al.,

; Strachan

et al.,

), the fusion of Gq or G

enabled the detection of the two

affinity sites in response to UCN3 binding. Whereas the lower affinity

binding sites (i.e., K

) of CRF

receptor-Gq and CRF

receptor-G

indicated the binding of the ligand to the receptor alone, the higher

affinity binding sites (i.e., K

) resembled the binding of UCN3 to the

CRF

receptor-Gq or CRF

receptor-G

complexes. The measured

values of UCN3 on CRF

receptor-Gq and CRF

receptor-G

were 59 ± 4.57 and 59 ± 19.8 nM, indicating binding affinity towards

the CRF

receptor receptor alone. In contrast, the measured K

values for the CRF

receptor-Gq and CRF

receptor-G

complexes

were 2.1 ± 0.29 and 1.0 ± 0.75 nM, respectively (Figure

4e,f

). Notably,

the

calculated

ratios,

which

closely represented the

net allosteric effect on the biased property, were approximately

2-fold higher for the CRF

receptor-G

chimera than for the CRF

receptor-Gq chimera. These results suggested that UCN3 had a more

biased preference for G

coupling than for Gq. Moreover, G

was

more enriched in pancreatic

Sst

-pm-labelled pancreatic

cells than in

other pancreatic cells (Figure

). Collectively, these results suggest

F I G U R E 3

The mechanism of Gs mediated signal pathway contributing to the secretion of somatostatin (SST) induced by UCN3. (a) Real-time

cAMP levels in response to UCN3 stimulation at different UCN3 concentrations were detected in CRF

receptor-overexpressing HEK293A cells

using cAMP-GloSensor Assay. The data are from three independent experiments (n

3). (b) Concentration-dependent response curves of CRF

receptor stimulated with UCN3 on intracellular cAMP accumulation. The data are from four independent experiments (n

4). (c,d) Time course

assay (c) and dose course assay (d) of CREB phosphorylation in response to UCN3 stimulation in TGP52 cells were conducted and detected using
western blot. In the time course assay, TGP52 cells were treated with 100 nM for indicated time (0, 2, 5, 10, 30 and 60 min). In the dose course
assay, TGP52 cells were treated with indicated concentrations of UCN3 for 30 min. Cells were then harvested for western blot analysis.

Representative western blot bands were shown in Figure

S3a,b

. Bands intensities were evaluated by Image J software and quantified. The data

are from three independent experiments (n

3). (e) Somatostatin secretion in response to UCN3, with or without Rp-cAMPS, H89 or NF449

were detected in mice islets. Islets diameters with radius ranging from 50

–

150

m were selected. The 250 islets from 5

–

8 mice were grouped

from WT (50 islets per group). The data are from three independent experiments (n

3). Mouse islets were isolated and treated with UCN3

(100 nM) alone or along with Rp-cAMPS (50

M), H89 (500 nM, PKA inhibitor), NF449 (1

M) for 1 h. Islets treated with vehicle were used as a

control. (f) SST secretion from islets isolated from WT mice or

Sst-Cre

fl/fl

mice in response to stimulation with UCN3, ryanodine or Rp-

cAMPS. Islets diameters with radius ranging from 50

–

150

m were selected. The 200 islets from 4

–

6 WT or

Sst-Cre

fl/fl

mice treated with

UCN3, ryanodine or Rp-cAMPS were grouped (50 islets per group). The data are from three independent experiments (n

3). (g) Somatostatin

secretion in response to UCN3 stimulation were detected in TGP52 cells with

Ryr1

Ryr2

Ryr3

knocking down. The data are from three

independent experiments (n

3). (h) Phosphorylation of RyR3 in response to UCN3 stimulation were examined in time-dependent manner using

western blot. TGP52 cells were prepared and treated with UCN3 (100 nM) for different time (0, 2, 5, 10 and 30 min). Then, RyR3 were
immunoprecipitated using anti-RyR3 and the phosphorylation of RyR3 by PKA were examined with anti-phospho-PKA-substrate antibody. The
data are from three independent experiments (n

3). (i

–

k) Phosphorylation of RyR3 fragments in response to UCN3 stimulation in HEK293 cells

with CRF2 receptor and different

Ryr3

constructs (RyR3-1-543 [i], RyR3-2616-2820 [j] or RyR3-4592-4868 [k]) overexpression. Different

Ryr3

constructs overexpressing RyR3-1-543 (i), RyR3-2616-2820 (j) or RyR3-4592-4868 (k) were cloned and co-transfected with CRF

receptors in

HEK293 cells. Cells were then treated with UCN3 (100 nM) in a time-dependent manner (0, 2, 5, 10, 30 min). RyR3 were immunoprecipitated
using anti-RyR3 and the phosphorylation of RyR3 by PKA were detected with anti-phospho-PKA-substrate antibody. Representative WB bands
were shown in Figure

S3g

. Bands intensities were evaluated using Image J software and quantified. The data are from three independent

experiments (n

3). (l) Phosphorylation of RyR3 fragments in response to UCN3 stimulation in HEK293 cells with different

Ryr3

constructs

overexpression. S156A, S160A, S2706A, S4697A or T4712A were introduced into

Ryr3

constructs overexpressing RyR3-1-543,

RyR3-2616-2820 or RyR3-4592-4868 and transfected in HEK293 cells. Cells were then treated with UCN3 (100 nM) for time accordingly to
results shown in Figure

, at which time phosphorylation of RyR3 reached to peak. RyR3 were immunoprecipitated using anti-RyR3 and the

phosphorylation of RyR3 by PKA were detected with anti-phospho-PKA-substrate antibody. Representative WB bands were shown in this figure.
Bands intensities were evaluated using Image J Software and quantified and shown in Figure

S3h

. (m) Graphic model showed the somatostatin

secretion from islet

cells was regulated by UCN3-CRF

receptor-Gs-RyR3 pathway. Statistical tests were not carried out where n was less

than 5.

WANG

ET AL

that the enrichment of the G

protein and the more biased property

of UCN3 towards G

than Gq may enhance the contributions of G

signalling to UCN3-induced SST secretion in

cells.

To provide further insights into the high G

expression in pan-

creatic

cells, we measured the mRNA levels of potential transcrip-

tion factors predicted to bind to G

promoters in

Sst

-pm-labelled

cells and compared them with those in other pancreatic cells

(DiGruccio et al.,

2016

; Segerstolpe et al.,

2016

). Specifically, the

mRNA levels of

Stat6

Foxo3

and

Tcf3

were more enriched in

cells

than in non-

islet cells (Figure

). Specifically, knockdown of

Stat6

but not the

Foxo3

Tcf3

, decreased the mRNA and protein levels of

(Figures

4h,i

and

). We then performed a quantitative ChIP

assay to profile the binding patterns of STAT6 across the region

encompassing approximately 2 kb upstream and downstream around

the G

protein transcription start sites (Figures

and

S4f

). The

results indicated that STAT6 directly bound to the DNA region

1219-843 downstream of the transcription initiation sites of the G

protein. These results suggested that the transcription factor STAT6,

which is more enriched in pancreatic

cells than in other cells, con-

tributed to G

expression and G

-mediated UCN3 effects on SST

secretion (Figure

3.5

-Arrestin1-mediated signalling pathways

contribute to the secretion of somatostatin (SST)
induced by UCN3

In addition to desensitizing G protein activation, arrestins also initiate

G protein-independent signalling downstream of a particular GPCR,

such as ther AT

receptor and adrenoceptors (Dong et al.,

; Fu

et al.,

; He et al.,

; Liu et al.,

; Wang et al.,

; Yang

et al.,

). Previously, we identified that

-arrestin1 played critical

roles in promoting

Sst

transcription by activating the ERK-PAX6 path-

way downstream of

-adrenergic receptor activation in pancreatic

cells (Li et al.,

); however, how arrestins participate in SST secre-

tion in pancreatic

cells has not been investigated. Here, results from

our animal models suggested that

-arrestin1 contributed to

UCN3-mediated SST secretion in pancreatic

cells; therefore, we

monitored the interaction between CRF

receptor and

-arrestin1 in

HEK293 cells overexpressing fluorescently tagged CRF

receptor and

-arrestin1. Without UCN3 stimulation, the CRF

receptor was mainly

located at the plasma membrane, whereas

-arrestin1 was evenly dis-

tributed in the cytoplasm (Figure

). After 5 min of UCN3 treatment,

the CRF

receptor and

-arrestin1 were colocalized in the plasma

membrane (punctate regions). We then measured the BRET signal

between the CRF

receptor-YFP and

-arrestin1-Rluc. The time

course and concentration-dependent curves (EC50: 91.3 ± 2.7 nM)

supported the interaction between the CRF

receptor and

-arrestin1,

even in the presence of low concentrations of UCN3, and the highest

BRET ratio over a 1-h duration was found at 5 min (Figure

5b,c

These data indicate that the

-arrestin1 signalling pathway plays an

essential role in its quick response under stimulation with physiologi-

cal concentrations of UCN3.

In TGP52 cells, the knockdown of

-arrestin1 further inhibited

UCN3-induced SST secretion following the administration of Rp-

cAMPS and U73122, inhibitors of cAMP and PLC, respectively

(Figure

). This result suggested that

-arrestin1 mediated cAMP/

Gs- and PLC/G

-independent SST secretion pathways in pancreatic

cells. In addition, the presence of the calcium chelator ethylene gly-

col tetraacetic acid (EGTA) did not phenocopy the effects of siArrb1,

suggesting that

-arrestin1 does not promote SST secretion by regu-

lating extracellular calcium influx.

Synaptotagmin, a family of synaptic vesicle proteins involved in

the

exocytosis

panel

hormones,

may

involved

in UCN3-induced SST secretion; however, this phenomenon has not

been characterized in pancreatic

cells. We therefore measured and

compared the mRNA levels of synaptotagmin protein subtypes in

UCN3-stimulated

cells and brain tissue (Figures

and

S5a

). Inter-

estingly, synaptotagmin-1 (

Syt1

)

, Syt2, Syt7, Syt9, Syt13, Syt14, Syt15

and

Syt16

were more enriched in pancreatic

cells. We therefore

knocked down each specific

Syt

subtype to determine which subtype

contributed to SST secretion in response to UCN3 stimulation after

the application of U73112 and Rp-cAMPS to compare these effects

with those of

Arrb1

knockdown (Figures

and

S5a

). The results indi-

cated that knocking down

Syt1

mostly mimicked the effects of

Arrb1

knockdown. Moreover,

-arrestin1 colocalized with SYT1 in HEK293

cells overexpressing CRF

receptors in response to UCN3 stimulation

(Figure

). The UCN3-induced association of

-arrestin1 with SYT1

was further supported by BRET and co-IP assays (Figure

5h,i

Together, these results suggested that the CRF

receptor-

-arrestin1

pathway contributed to SST secretion in pancreatic

cells by recruit-

ing SYT1 to

-arrestin1 and activating the corresponding exocytosis

pathway (Figure

3.6

Long-term exposure to different levels of

UCN3 showed distinct effects

In addition to acute responses, we next examined how long-term

exposure to UCN3 at different concentrations modulates islet func-

tions. Importantly, the glucose-induced insulin secretion level after

long-term exposure (72 h) to 100-nM UCN3 in mouse islets was

decreased compared with that of the control (treatment with

CYN154806, a somatostatin receptor antagonist) (Figure

), and this

is consistent with the conclusion that SST of islet

cells can exert

tetanic inhibitory effects on insulin and glucagon secretion (Hauge-

Evans et al.,

2009

). In contrast, long-term exposure (72 h) to 10-nM

UCN3 showed no effects on glucose-induced insulin secretion. We

then examined changes in the mRNA levels of several genes related

to glucose-stimulated insulin secretion (Figure

). The mRNA levels

Adcy8

and

Glp1r

were decreased after long-term exposure to either

10- or 100-nM UCN3. Interestingly, whereas the mRNA level of

Glut2

decreased in response to 100-nM UCN3 exposure, its level increased

after 10-nM UCN3 exposure. We noticed that 100-nM UCN3 specifi-

cally activated G

signalling in pancreatic

cells (compared to 10-nM

UCN3 treatment). We therefore specifically knocked down G

WANG

ET AL

F I G U R E 4

The mechanism of the G

/Gq mediated signal pathway contributing to the secretion of somatostatin (SST) induced by UCN3.

(a) Somatostatin secretion in response to UCN3 stimulation were detected in TGP52 cells with

Gnaq

(Gq),

Gna11

(G11) or

Arrb1

knocking down.

TGP52 cells were transfected with siRNAs targeted to

Gnaq

Gna11

Arrb1

for 48 h and then treated with UCN3 (10 nM), U73122 (10

M, PLC

inhibitor) or NF449 (1

M) for 1 h. The data are from three independent experiments (n

3). (b) BRET-based G protein trimer dissociation assays

were used to determine the effects of UCN3 on G

coupling to CRF

receptors in a concentration-dependent manner. The data are from three

independent experiments (n

3). (c) BRET-based G protein trimer dissociation assays were used to determine the effects of UCN3 on G11

coupling of CRF

receptor in a time-dependent manner (0, 1, 2, 5, 10, 30 and 60 min). The data are from three independent experiments (n

3).

(d) Relative mRNA levels of

Gnaq

(Gq) and

Gna11

) in islet

cells and non-

cells were detected by qRT-PCR. Islet

cells were labelled with

RFP in

Sst-Cre

Rosa

lsl-RFP

mice. Approximately 1 * 10

RFP

or RFP

cells were collected from 3

–

Sst-Cre

Rosa

lsl-RFP

mice by flow cytometry

and were prepared for qRT-PCR detection. The data are from three independent experiments (n

3). (e,f) Competitive binding curve of UCN3 in

CRF

receptor-G

q (e), CRF

receptor-G

(f) or CRF

receptor transfected HEK293 cells. The data are from three independent experiments

3). (g) Relative mRNA levels of potential transcription factors (

Arnt

Foxa2

Foxo1

Foxo3

Klf4

Nfatc2

Stat6

Tcfl2

Tcf3)

binding to

Gna11

promoters in islet

cells and non-

cells were detected by qRT-PCR. Islet

cells were labelled with RFP in

Sst-Cre

Rosa

lsl-RFP

mice.

Approximately 1 * 10

RFP

or RFP

cells were collected from 3

–

Sst-Cre

Rosa

lsl-RFP

mice by flow cytometry and were prepared for qRT-PCR

detection. The data are from three independent experiments (n

3). (h,i) The relative expression levels of G

in response to

Foxo3

Stat6

Tcf3

knocking down were evaluated in TGP52 cells. TGP52 cells were transfected with siRNAs targeted to

Foxo3

Stat6

, or

Tcf3

for 48 hours. Cells

were then harvested for qRT-PCR and western blot analysis. Relative mRNA levels of

Foxo3

Stat6

Tcf3

Gna11

(

) were analysed by qRT-

PCR (h and Figure

S4d

). Representative immunoblot bands showing G11 protein levels were shown in Figure

S4e

. Band intensities were

evaluated using Image J and analysed in (i). The data are from three independent experiments (n

3). (j) ChIP analysis of STAT6 binding patterns

Gna11

(

G11

) promoter region in TGP52 cells. STAT6 were overexpressed in TGP52 cells. After then, cells were cross-linked, sonicated, and

harvested for immunoprecipitation using anti-STAT6 antibody. G11 promoter fragments were detected using RT-PCR and analysed.
Representative DNA bands were shown in Figure

S4f

. The data are from three independent experiments (n

3). (k) Graphic model showed the

somatostatin secretion from islet

cells was regulated by STAT6-G11 pathway Statistical tests were not carried out where n was less than 5.

WANG

ET AL

control group. We performed immunofluorescence experiments to

monitor the expression of MKI67 in mouse islets after 72 h of expo-

sure to 10- or 100-nM UCN3 (Figure

). The data revealed that

10-nM UCN3 increased

Mki67

expression in pancreatic islets,

whereas 100-nM UCN3 did not.

D I S C U S S I O N

Islets are complex microorgans that consist of several types of endo-

crine cell that play a key role in the regulation of whole-body energy

metabolism (Rorsman & Huising,

). Specific paracrine interac-

tions among these islet cells are critical in mediating appropriate

responses to different physiological contexts (Arrojo e Drigo

et al.,

2019

; Unger & Orci,

2010

; van der Meulen et al.,

). As an

important paracrine factor, UCN3 potentiates high glucose-induced

SST secretion to promote a negative feedback loop that attenuates

insulin release (Li et al.,

; van der Meulen et al.,

). In

response to endogenous UCN3 stimulation, the CRF

receptor may

couple to different downstream effectors, such as Gs, Gq and arrest-

ins. How these different signals are present in pancreatic

cells has

not been previously investigated. Here, our results suggested that at

least three direct downstream effectors, including Gs, G

and

-arrestin1, directly contributed to UCN3-induced CRF

receptor in

pancreatic

cells to modulate acute SST secretion. While RyR3

phosphorylation by PKA may play important roles in Gs-mediated

UCN3-CRF

receptor function in SST secretion, its interaction with

Syt1 may contribute to

-arrestin1-mediated CRF

receptor function

in pancreatic

cells. Moreover, the specific expression of stat6 may

contribute to G

expression and G

-mediated UCN3 effects on

SST in pancreatic

cells. Therefore, our study provides a preliminary

landscape of different downstream signalling pathways that partici-

pate in UCN3-CRF

receptor axis-mediated SST secretion in pancre-

atic

cells. Interestingly, we found UCN3 has preferential signalling

at different concentrations. The 10 nM UCN3 mainly activates the

Gs and

-arrestin1 signalling downstream CRF

receptors in

cells of

pancreatic

islets.

These

two

pathways

also

contributed

UCN3-induced SST secretion in pancreatic

-cells, which was previ-

ously unknown (Figures

and

S2c

). Moreover, a high concentration

(100-nM UCN3), in addition to activating the Gs and

-arrestin1 sig-

nalling pathways, could also activate G

to promote SST secretion

through the PLC-calcium signalling pathway. The important func-

tional roles of G

and how its expression is modulated in pancreatic

cell have never been explored in previous studies. Our findings

provide a deeper understanding of the UCN3-CRF

receptor-SST

paracrine signalling axis and how SST secretion is regulated by

pancreatic

cells.

Another notable finding is that high and low concentrations of

UCN3 have different kinds of effects on islet cells. In a longer time

window (up to 3 days; compared to 1 h), 10- and 100-nM UCN3

F I G U R E 5

The

-arrestin1 mediated signal pathway contributes to the secretion of somatostatin (SST) induced by UCN3. (a) Colocalization of

-arrestin1 and the CRF

receptor in response to UCN3 stimulation was detected in HEK293 cells. CRF

receptor-YFP and

-arrestin1-RFP were

overexpressed in HEK293 cells and stimulated with UCN3 (100 nM) for 5 min. Analysis of co-localization for

-arrestin1 and the CRF

receptor

was conducted using immunofluorescence. Representative images were shown here. Scar bar: 20

m. Three cell slides were performed for an

independent experiment; 4

–

6 random areas were selected from every cell slide. Data were from three independent experiments (n

3). Analysis

of co-localization of the CRF

receptor and

-arrestin1 of HEK293 cells stimulated with UCN3 (100 nM) for 0 and 5 min, CRF

receptor and

-arrestin1 fluorescence intensities was analysed by Pearson's correlation analysis. The Pearson's correlation coefficient of co-localization of

CRF

receptor and

-arrestin1 of HEK293 cells without stimulation or stimulated with UCN3 (100 nM) for 5 min were

0.15 and 0.83,

respectively. (b,c) BRET-based

-arrestin1 recruitment assays were used to determine the effects of UCN3 on

-arrestin1 coupling to CRF

receptor as time-dependent (b) or dose-dependent (c) manner. BRET ratio at different time were compared to that at zero time. The data are from
three independent experiments (n

3). (d) Somatostatin secretion in response to different pharmacological interventions was detected in TGP52.

TGP52 cells were transfected with siRNAs targeted to

Arrb1

or siCon for 48 h and then treated with UCN3 (10 nM), Rp-cAMPS (50

M), U73122

(10

M) or EGTA for 1 h. The data are from three independent experiments (n

3). (e) The relative expression levels of selected Synaptotagmins

in SST-labelled islet

cells were examined by qRT-PCR. Islet

cells were labelled with RFP in

Sst-Cre

Rosa

lsl-RFP

mice. Approximately 1 * 10

RFP

cells were collected from 3

–

Sst-Cre

Rosa

lsl-RFP

mice by flow cytometry and prepared for qRT-PCR detection using brain tissue as a

control. The data are from three independent experiments (n

3). (f) Co-localization of

-arrestin1 and SYT1 in response to UCN3 stimulation

was detected in HEK293 cells. Representative images were shown here. Scar bar: 20

m. Three cell slides were performed for an independent

experiment; 4

–

6 random areas were selected from every cell slide. Data were from three independent experiments (n

3). Analysis of co-

localization SYT1 and

-arrestin1 of HEK293 cells stimulated with UCN3 (100 nM) for 0 and 20 min, SYT1 and

-arrestin1 fluorescence

intensities were analysed by Pearson's correlation analysis. The Pearson's correlation coefficient of co-localization of SYT1 and

-arrestin1 of

HEK293 cells without stimulation or stimulated with UCN3 (100 nM) for 20 min was 0.28 and 0.6, respectively. (g) Somatostatin secretion in
response to different pharmacological intervention were detected in TGP52 cells. TGP52 cells were transfected with siRNAs targeted to

Arrb1

different

Synaptotagmins

for 48 h and then treated with UCN3 (10 nM), with or without Rp-cAMPS (50

M), or U73122 (10

M) for 1 h. The

data are from three independent experiments (n

3). (h,i) Interaction of

-arrestin1 and Syt1 in response to UCN3 stimulation were detected

using BRET (h) and co-IP (i).

-arrestin1-Rluc and Syt1-YFP tags were co-transfected in HEK293 cells for 48 h in BRET assay, while

-arrestin1

and Syt1 fused with HA or MYC were co-transfected in HEK293 cells for 48 h in co-IP assay. Cells were then harvested after UCN3 stimulation
and prepared for indicated experiments following protocols. Dose-dependent interactions of two proteins upon UCN3 stimulation were shown in
(h). Representative co-IP bands were shown in (i). The data are from three independent experiments (n

3). (j) Graphic model showed the

somatostatin secretion from islet

cells was regulated by

-arrestin1-SYT1 pathway. Statistical tests were not carried out where n was less

than 5.

WANG

ET AL

F I G U R E 6

Different gene transcriptional regulatory pathways in

cell related with UNC3-CRF2 receptor signal pathway contribute to insulin

secretion. (a) Insulin secretion from islets isolated from WT mice in response to stimulation with UCN3 or CYN154806. Islet diameters with
radius ranging from 50

–

150

m were selected. The 600 islets from 12

–

16 WT mice treated with UCN3 or CYN154806 were grouped (50 islets

per group). The data are from three independent experiments (n

3). (b) Heat map of

cell markers in islets stimulated with UCN3 or

CYN154806. Red and blue blocks represented upregulation or downregulation of specific genes in response to the stimulation. The 400 hundred
islets from 8

–

12 mice were grouped from WT mice (100 islets per group). The data are from three independent experiments (n

3). (c) Insulin

secretion in response to long-time UCN3 or CYN154806 stimulation were detected in

Sst-Cre

mice islets infected with lentivirus encoding

G11-shRNA-GFP

fl/fl

. Islets diameters with radius ranging from 50

–

150

m were selected. Two hundred islets from 4

–

8 mice were grouped from

Sst-Cre

control-shRNA-GFP

fl/fl

and

Sst-Cre

G11-shRNA-GFP

fl/fl

mice (100 islets per group). Islets were isolated and stimulated with UCN3

(100 nM) or CYN154806 for 72 h. Insulin secretion were measured using ELISA kit in response to glucose (1 or 20 nM) treatment. The data are
from three independent experiments (n

3). (d) The relative expression levels of selected

cell markers in

Sst-Cre

mice islets infected with

lentivirus encoding G11-shRNA-GFP

fl/fl

. Two hundred islets from 4

–

8 mice were grouped from

Sst-Cre

control-shRNA-GFP

fl/fl

and

Sst-

Cre

G11-shRNA-GFP

fl/fl

mice (100 islets per group). The data are from three independent experiments (n

3). (e) The relative expression

levels of cell cycle markers in islets stimulated with UCN3 for different concentrations were detected using qRT-PCR. Three hundred islets from
6

–

8 mice were grouped from WT mice (100 islets per group). The data are from three independent experiments (n

3). (f) Immunostaining (left)

and quantification (right) analysis of insulin and mki67 in islets were conducted in response to UCN3 stimulation for different concentrations.
Representative images were shown here. Scale bar: 100

m. Mki67

cells were calculated and analysed. In every independent experiment,

200 islets were stimulated with UCN3 in every group and prepared for immunostaining. The data are from six independent experiments (n

6).

Statistical tests were not carried out where n was less than 5.

WANG

ET AL

have opposite effects on insulin secretion through CRF

receptor sig-

nalling involved in

cell adaptive responses. In mouse

cells,

Glut2

is established as the primary glucose transporter and modulates insu-

lin secretion (Guillam et al.,

2000

; Low et al.,

; Thorens

et al.,

2000

). Our studies suggest that the expression levels of

Glut2

are closely regulated by UCN3. The change in the mRNA levels in

cells and the functional/experimental data from G

KO mouse islets

showed that

Glut2

was down-regulated in pancreatic islets in

response to a high concentration of UCN3. In contrast, 10-nM UCN3

induced increased expression of the typical pancreatic

cell tran-

scription factors

Pdx1

and

Nkx6.1

and the proliferation markers

Ki67,

Ccnd1, Ccnd2

and

Cdk2

(Cogger et al.,

; Lin et al.,

; Russ

et al.,

Immunoblotting and Immunochemistry

) compared with the control group without UCN3 stimula-

tion. These results suggested that low concentrations of UCN3,

which may mainly activate Gs and

-arrestin1 signalling, had benefi-

cial effects on glucose homeostasis. In contrast, the overactivation of

CRF

receptor-G

signalling by high concentrations of UCN3 may

have detrimental effects on insulin secretion by downregulating

Glut2

expression levels.

In summary, we have provided a landscape of different G protein

or arrestin subtypes in pancreatic

cells, as well as their key down-

stream signalling hubs, that participate in UCN3-CRF

receptor-

mediated SST secretion in the islet circuit of crosstalk between islet

and

cells. The differential effects of different UCN3 levels and the

contribution of different G protein subtypes or arrestins to islet func-

tion may help us to further understand glucose homeostasis through

hormone receptor interaction networks.

A U T H O R C O N T R I B U T I O N S

J.-P. S. and X. Y. initiated the research. J. W., J.-P. S. and X. Y.

designed and supervised the overall project. X. Y. conceived the

method to identify signalling pathway and function of CRF2 receptor

in response to UCN3 stimulation. J.-P. S. and X. Y. designed all func-

tional assays. H. Q. and J. L. performed G protein screening in

Sst

-pm-

labelled islet cells. K. W., M. G., H. Q., J. L. W. P. and Q. C. isolated

mice islets and performed ex vivo experiments. K. W., M. G., W. A.

and J. L. performed cell experiments, immunofluorescence studies and

ligand binding experiments. K. W., M. G., J. L. and S. N. performed

BRET and western blot experiments. K. W., M. G., H. Q., W. A., J. L.,

S. N., F. Y., P. X., J. C., L. F., J. W., J.-P. S. and X. Y. participated in data

analysis and interpretation. K. W., M. G., W. A., J. L., S. N., F. Y., P. X.,

L. F., J. W., J.-P. S. and X. Y. wrote and revised the manuscript. All the

authors have seen and commented on the manuscript.

A C K N O W L E D G E M E N T S

This work was supported by the National Science Fund for Distin-

guished Young Scholars Grant (81825022 to J.-P. S., 82225011 to X.

Y.), Key Research Project of the Natural Science Foundation of

Beijing (Z200019 to J.-P. S.), National Natural Science Foundation of

China Grant (31971195 to P.X., 92057121 to X.Y., 32130055 to J.-P.

S., 91939301 to J.-P.S., 82300890 to J.C.), the Major Fundamental

Research Program of Natural Science Foundation of Shandong Prov-

ince, China (ZR2021ZD18 to X.Y.), China National Postdoctoral

Program for Innovative Talents (BX20230201 to J.C.), China Postdoc-

toral Science Foundation (2023M742066 to J.C.), the Natural Science

Foundation of Shandong Province, China (ZR2023MH196 to S.-L.N.),

and National Key Research and Development Program of China

(2019YFA0904200 to J.-P. S.).

C O N F L I C T O F I N T E R E S T S T A T E M E N T

All authors declare no competing interests.

D A T A A V A I L A B I L I T Y S T A T E M E N T

The data that support the findings of this study are available from the

corresponding author upon reasonable request. Some data may not

be made available because of privacy or ethical restrictions.

D E C L A R A T I O N O F T R A N S P A R E N C Y A N D S C I E N T I F I C

R I G O U R

This Declaration acknowledges that this paper adheres to the princi-

ples for transparent reporting and scientific rigour of preclinical

research as stated in the

BJP

guidelines for

Natural Products

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Design and Analysis

and

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