(AFM) based force spectroscopy on model human proximal tubule
cells (HK2) Æ TGF-b1 (10ng/ml) at 37°C. Fitted with a spherical
bead, AFM cantilevers functioning under constant force mode
measured mechanical forces over time and viscoelastic properties
were calculated using the Maxwell model of viscoelasticity.
Results: Data suggest that both control and TGF-b1-treated cells
exhibit transient (5s) force relaxation of 0.7 and 0.3nN, respec-
tively, in response to physical deformation. The initial phase was
followed by stabilisation in force over a subsequent 30-s period.
TGF-b1-treated cells demonstrated a reduction in relaxation
characteristics, suggesting that complex viscoelastic components
are strongly affected by reorganisation of the actin cytoskeleton.
Data indicate that viscosities after treatment vary up to 35% (12
cells, n = 3, p < 0.001). Treated cells showed a threefold reduction
in experimental decay of the force time curve that leads to
signiﬁcant changes in the adhesive deformation behaviour.
Conclusion: TGF-b1 (10ng/ml) triggered complex nanomechani-
cal changes in the viscous-elastic behaviour of single cells. Our
research suggests that the progression of the disease instigates
intricate physical changes that may, in part, mediate altered cell–
extracellular matrix (ECM) interactions linked to altered cell
phenotype in tubular injury.
Basic and clinical science posters: Beta cells, islets and
Identiﬁcation of pro-apoptotic signalling
through the GPRC5B receptor in beta cells
by CRISPR-Cas9-mediated genome editing
P ATANES, I Ruz-Maldonado, R Hawkes, B Liu, S Amisten and
Department of Diabetes, Faculty of Life Sciences & Medicine, King’s College
London, London, UK
Background/Aims: GPRC5B is an orphan receptor that is highly
expressed in human and mouse islets, and we have previously
reported that its down-regulation in islets protects against apoptosis.
As there are no agonists identiﬁed for GPRC5B, we investigated its
role in MIN6 beta cells by stably deleting it using the CRISPR-Cas9
technology, then re-introducing it in a graded fashion.
Methods: Efﬁciency of GFP plasmid delivery into MIN6 beta
cells was determined by cell sorting following transfection with
eight reagents. CRISPR-Cas9 was used to stably delete the Gprc5b
gene in MIN6 beta cells, and 0.01% to 100% Gprc5b plasmid was
introduced by transient transfection to restore Gprc5b expression.
Standard techniques were used to quantify Gprc5b mRNA
expression, apoptosis, and signalling pathways downstream of
GPRC5B were identiﬁed using Cignal 45-Pathway Reporter Arrays
and western blotting.
provided the best MIN6 beta cell transfec-
tion efﬁciency (29 Æ 0.5% GFP-positive cells). Isolation of Gprc5b
KO cell clones using CRISPR-Cas9 was established, and DNA
sequencing conﬁrmed deletion of Gprc5b nucleotides by this
strategy. Re-expression of GPRC5B in Gprc5b KO MIN6 beta
cells increased apoptosis (119.5 Æ 4% of maximum cytokine
response, p < 0.01) and induced up-regulation of transforming
growth factor (TGF)-b1 and Interferon gamma (IFNc) pro-
apoptotic pathways (3.4 Æ 0.9% and 2.9 Æ 0.3 log2 fold change,
respectively). Activation of pro-apoptotic signalling was conﬁrmed
by western blotting, which indicated signiﬁcant (p < 0.001)
GPRC5B-induced phosphorylation of AKT (+125 Æ 7.6%) and
STAT1 (+185 Æ 14.0%).
Conclusion: We have developed an efﬁcacious CRISPR-Cas9 gene
editing protocol, which we used to delete the Gprc5b gene in
MIN6 beta cells. Re-introduction of GPRC5B expression allowed
us to identify signalling pathways linking GPRC5B with beta cell
Roles for the Type 2 diabetes-associated
genes C2CD4A and C2CD4B in the control of
N MOUSAVY GHARAVY, X Li, I Leclerc, A Martinez-Sanchez
and GA Rutter
Medicine, Imperial College London, London, UK
Background: Single-nucleotide polymorphisms near C2CD4A
and C2CD4B are associated with altered pro-insulin levels and
Type 2 diabetes risk at genome-wide signiﬁcance. Altered expres-
sion of both C2CD4A and C2CD4B has been reported in islets in
association with risk variants. Both genes encode Ca
proteins thought to be localised to the nucleus. Here, we address
their potential role in glucose homeostasis in vivo and in vitro.
Methods: C2CD4B null mice were generated by the International
Mouse Phenotyping Consortium using CRISPR/Cas9-mediated
genome editing. Intraperitoneal glucose tolerance tests (IPGTTs)
were performed using standard protocols. Subcellular analysis of
ﬂag-tagged constructs was performed by immunocytochemistry
and confocal ﬂuorescence microscopy.
Results: Animals deleted globally for C2cd4b showed mild
dysglycaemia (females at 12 weeks: 15min IPGTT: wild type
(WT), 11.5 Æ 0.85 mmol/l; knockout (KO) 14.78 Æ 1.05 mmol/l,
n = 7 to 11, p = 0.0129; males at 16 weeks, WT,
13.38 Æ 0.67 mmol/l; KO, 16.00 Æ 0.56 mmol/l, n = 8,
p = 0.045). In human-derived EndoC BH1 beta cells, C2CD4A
and C2CD4B were localised to the cytoplasm and nucleus in
92.31 Æ 3.3% and 79.22 Æ 10.12% of cells, respectively
(n = 100 and 80 cells). Additional localisation to the plasma
membrane was observed in 3.64 Æ 3.6% and C2CD4B
DIABETICMedicine Abstracts of the Diabetes UK Professional Conference, 14-16 March 2018
ª 2018 The Authors, presented at the Diabetes UK Professional Conference ª 2018 Diabetes UK. Diabetic Medicine, 35 (Suppl. 1), 36–205