Diabetes Research and Clinical Practice - Volume 120 - Supplement 1 - October 2016

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decrease in Dbp mRNA and a marked increase in Nfil3/E4bp4
mRNA. Meanwhile, Chop mRNA increased 24-fold as com-
pared with the level in controls. Isolated islets treated with
0.5 µM TG for 24 h or 2 µg/mL tunicamycin (TM) showed very
similar changes in the expressions of clock genes and Ddit3.
Overall, chronic ER stress should decreases DBP transcriptional
activity in β-cells. To explore the role of DBP transcriptional
activity in ER stress-induced β-cell dysfunction, we created
transgenic mice expressing E4BP4 under the control of the
mouse insulin I gene promoter (MIP), in which E4BP4 should β-
cell-specifically compete for D-box, with DBP and suppress the
expressions of the targets of DBP. We analyzed insulin
secretion in the perfused pancreas. MIP-E4BP4 mice had
markedly reduced and delayed insulin secretion during
perfusion with 16.8 mM glucose. In pathophysiological set-
tings, our data indicate that chronic ER stress disrupts this
circadian alignment leading to β-cell failure. Elucidating the
role of circadian clocks in ER stress-induced β-cell failure could
introduce novel approaches to treating diabetes.
OL06-2
Effect of heparan sulfate proteoglycan Syndecan-4 on the
insulin secretory response
Iwao TAKAHASHI 1, Shuhei YAMADA2, Koji NATA1. 1Department
of Medical Biochemistry, School of Pharmacy, Iwate Medical
University, Yahaba, 2Department of Pathobiochemistry, Faculty of
Pharmacy, Meijo University, Nagoya, Japan
Objectives: Heparan sulfate (HS) proteoglycans (PGs) comprise
a core protein to which extracellular glycosaminoglycan
chains are attached. We recently found that HS is localized
exclusively around β-cells in the islets of adult mice and is
required for islet morphogenesis, β-cell proliferation and
insulin secretion. Furthermore, we found that the 3- O-sulfate
groups of HS are necessary for maintaining glucose-induced
insulin secretion (GIIS). So far it is not known, however, which
core proteins are crucial in this process and how they function
to modulate β-cell function. The aim of this study was to
identify the core protein(s) which are critical for insulin
secretion, and to clarify the effect of the core protein(s) on
insulin secretion.
Methods: To investigate the participation of HSPGs in the
insulin secretion mechanism, MIN6 cells, a mouse pancreatic
β -cell line, were subcloned by limiting dilution method. The
subcloned MIN6 cells were selected based on their insulin
secretion level following stimulation with glucose or KCl.
Furthermore, we examined the expression of HS and core
proteins in the subclones. Using silencing and overexpression
of the core protein which involved in insulin secretion in
cultured subclones, we examined changes of disaccharide
composition of HS, GIIS and expression of the genes related to
insulin secretion.
Results: Syndecan-4 (Sdc4), one of the major HS-containing
core proteins, is distributed on the cell surface. The results
from our screening experiments indicated that only Sdc4-
expressing subclones are able to secrete insulin in response to
glucose. While, Sdc4-lacking subclones had significantly low
GIIS response and no HS from cell surface PGs. Silencing of
Sdc4 by RNA interference reduced GIIS by about 50% at 25 mM
glucose (p < 0.001), whereas the overexpression of Sdc4
increased the insulin secretory response by approximately 2-
to 8-fold as compared with control cells. Based on HPLC
analysis, the amount of HS had increased in Sdc4-overexpres-
sing cells by approx. 5.5- to 29-fold. In Sdc4-overexpressing
cells, Glut1 or Gck mRNA levels were elevated, suggesting that
these genes have enhancing effects on insulin secretion
pathway.
Conclusions: Our data indicate that the HSPG Sdc4 plays
important role(s) in the GIIS response of pancreatic β-cells.
However, there exists the differences in glucose responsive-
ness and expression levels of GIIS-related genes among the
Sdc4-overexpressing subclones. The further investigation
is necessary to clarify the mechanisms in the relationship
between Sdc4 and GIIS.
OL06-3
Elucidation of mechanism of human pancreatic beta-cell
maturation with hiPSC reporter line
Toshihiro NAKAMURA1, Junji FUJIKURA1, Yasushi KONDO1,
Ryo ITO1, Nobuya INAGAKI1 . 1Department of Diabetes,
Endocrinology and Nutrition, Kyoto University, Japan
After the discovery of human induced pluripotent stem cell
(hiPS), regenerative therapy with hiPSCs for patient with
insulin-dependent diabetes mellitus (IDDM) has been
expected, but there are still several tasks to clarify before we
perform transplantation of hiPSC-derived insulin secreting
cells in future.
One of the difficulties comes from unknown mechanism of
human pancreatic beta-cell maturation. The mechanism has
been clarified gradually as it has been reported that several
transcriptional factors play an important role in beta-cell
maturation such as Pdx1, MafA, NeuroD, Nkx6.1, GLP-1R and
Rfx6, but the difference between species has been pointed out
in some reports, so more accurate mechanism in human still
remains to be elucidated.
EGFP knock-in artificial gene, that express EGFP in a down-
stream of the promoter region of the target gene, enables us to
detect when it expresses and when it does not. Therefore, we
consider the reporter line is suitable to evaluate the function of
target gene.
Our group has already constructed Insulin(INS)-GFP reporter
line with this method and we are now establishing hiPSC-
reporter line of several transcriptional factors. Among the
transcriptional factors, MafA (V-maf musculoaponeurotic
fibrosarcoma oncogene homolog A) attracts our attention for
its expression synchronized with beta-cell maturation so that
we are working especially on MafA-EGFP reporter line.
Cloningmethod we use is called Red-ET recombination system
with bacterial artificial chromosome (BAC) which contains
target gene.
To validate the accuracy of the reporter line, we are planning to
perform in vitro and in vivo assay of the reporter line. As to in
vitro assay, we are going to check the marker of undifferentia-
tion and pluripotency and then follow several methods of hiPS
differentiation and evaluate when and how much the target
genes are expressed, and evaluate the ability of glucose-
sensitive insulin secretion (GSIS). In the point of in vivo assay,
we will transplant the undifferentiated cell colonies to mice to
make tumor with ectoderm, mesoderm and endoderm to
certify its pluripotency, and transplant semi-differentiated cell
colonies to trace its destination in vivo compared to in vitro,
and transplant differentiated cell colonies to check in vivo
function of GSIS. We are considering that this research can
contribute to more accurate hiPSC regenerative therapy in
future for IDDM patients. In this presentation, we will report
our latest findings of our reporter line.
OL06-4
The effect of caveolin-1 on the process of proliferation and
apoptosis and function regulation in beta cell
Haicheng LI1, Hangya PENG1, Haixia XU1, Fen XU1, Shuo LIN1,
Keyi LIN1, Wen ZENG1, Longyi ZENG1*. 1The 3rd Affiliated
Hospital Sun Yat-Sen University, China
Beta cell loss is a hallmark in all forms of diabetes mellitus.
Strategies to prevent beta cell loss and dysfunction are
urgently needed. Our study aims to access the influence of
caveolin-1 on the beta cell’s apoptosis and function. We
knockdown the expression level of caveolin-1 in both NIT-1
cell and islet isolated from C57BL/6J mice by RNA interference
technique which realized by transfer a shRNA vector target
Oral Presentations / Diabetes Research and Clinical Practice 120S1 (2016) S40–S64 S53