Page 65 - I. Chemical biology and drug discovery

DH047 induces insulin sensitivity and glucose uptake

via inhibition of protein tyrosine phosphatases
1
1
1
Dohee Ahn , Sun-Young Yoon , Ji Young Hwang and Sang J. Chung 1
1 College of Pharmacy, Sungkyunkwan University, 2066 Seoburo Jangangu, Suwon 16419, South Korea
BACKGROUND AIM
Type 2 diabetes mellitus (T2DM) is a disease To identify antidiabetic effect natural compound inhibitor for
characterized by insulin resistance. Insulin diabetes-related PTPs, we screened natural compound isolated
resistance has a feature of defects in insulin from a certain plant. Then, we examined IC 50 and cooperative
signaling and some function of PTPs have been
studied and proved to be involved in this signaling binding of potent inhibitor through evaluating hill coefficient in vitro.
pathway. We have purified 77 PTPs and screened In C2C12 muscle cells and 3T3-L1 adipocytes, we investigated this
natural compound isolated from a certain plant to inhibitor can increase GLUT4 translocation to the plasma membrane
identify potent inhibitors of PTPs involved in insulin protein and stimulates glucose uptake. Moreover, we studied
resistance. signaling pathway. In this study, we suggest DH002 is a potential
therapeutic candidate for T2DM.
METHODS

3T3-L1 C2C12
Compound adipocytes myotubes
DH002
GLUT4 translocation ↑
2-NBDG uptake ↑
P-AMPK ↑
P-AKT ↑

RESULTS

Figure 1. Enzyme purification. (A) (B) (C)
10% SDS-PAGE gel with
Coomassie-staining. PTPN1 (1),
PTPN2 (2), PTPN9 (3), PTPN11
(4), DUSP9 (5) cloned into
pET28a vector tagged with MBP
(n-terminal; PTPN2, DUSP9,
PTPN11) and polyhistidine (n-
terminal; PTPN1, PTPN9; c-
terminal; PTPN2, DUSP9,
PTPN11) was overexpressed in
E.coli and purified by affinity
chromatography. Figure 2. DH002 increased GLUT4 expression and translocation. (A) During the differentiation of C2C12 muscle cells, the cells were incubated
with 40 μM DH002. Then, the cells were stimulated with 100 nM insulin for 1 h. Nucleus and Glut4 were stained using immunocytochemistry (ICC).
The image shows DH002 increased GLUT4 expression and translocation. DH002 stimulates glucose uptake in (B) C2C12 and (C) 3T3-L1 cell line.
Cells were treated with 40 μM of DH002 and 100 nM insulin after differentiation, at day 4 (C2C12) or 7 (3T3-L1). Glucose uptake was measured
using 2-NBDG. Results are expressed as the mean ± standard deviation.
(A) (B) (C)

Table 1. Kinetic properties of PTPs Activity of PTPs were measured
using DIFMUP under pH 6.0 or pH 7.0 reaction buffer. Reaction buffer
includes 20 mM pH 6.0 bis-tris-HCl or pH 7.0 tris-HCl, 150 mM NaCl,
2.5 mM DTT, 0.01% triton X-100 Figure 3. DH002 stimulates AKT and
(D) (E)
AMPK phosphorylation of C2C12 muscle
cells and 3T3-L1 adipocyte cells (A, B, C)
Table 2. IC 50 determination of Effects of DH002 on insulin signaling
DH002 The table shows DH002 pathway and P-AMPK signaling pathway.
inhibits diabetes-related PTPs C2C12 myotubes (A) and 3T3-L1 cells (B, C)
with micromolar IC 50 and hill were treated with DH002 for 2 h (A) or 6 h
coefficient (n H ) greater than 1.0 (B, C) and stimulated with 100 nM insulin for
indicates DH002 binding for PTPs 30 min. (D, E) During the differentiation of
is positively cooperative. C2C12 myotubes (D) and 3T3-L1 cells (E),
the cells were incubated with 10, 40 μM
DH002 for 72 h.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
Several protein tyrosine phosphatases (PTPs) are involved in insulin 1. Y. Bu, T. Shi, M. Meng, G. Kong, Y. This research was supported by the Bio & Medical
signaling pathway and altered PTPs activity leads to T2D. In this study, Tian, Q. Chen, X. Yao, G. Feng, H. Technology Development Program of the National
screened fractions isolated from the certain plant shows over 70% Chen and Z. Lu, Bioorg Med Chem Lett Research Foundation (NRF) funded by the Korean
inhibition for SHP2 and among single compounds separated from the 2011, 21, 874-878. government (MSIT) (NRF-2012M3A9C4048775 and
fractions, 3 hit compounds were identified. DH002 inhibited 5 diabetes- NRF-2017M3A9C8031995)
related PTPs and IC 50 of DH002 for PTPs measured via enzyme kinetics 2. Mozzarelli, A., et al., J Biol Chem,
were micromolar range. Moreover, its antidiabetic effect was investigated 1996. 271(7): p. 3627-32. Contact information
in C2C12 and 3T3-L1 cell lines. DH002 stimulates glucose uptake via 3. Hsieh, C.T., et al., Int J Mol Sci,
increased GLUT4 expression and translocation in C2C12 and 3T3-L1 2018. 19(9).
cells. We investigated increased glucose uptake was due to AMPK and E-mail: sjchung@skku.edu
Akt signaling pathway. As a result, our findings suggest that these
inhibitors can be a potential candidate as an antidiabetic drug. However,
further studies are required to explain the antidiabetic effect of DH002.