Insulin-mimetic and anti-inflammatory potential of a vanadyl-Schiff base complex for its application against diabetes

Article abstract of DOI:10.1039/c6ra11111d

Insulin signalling causes the translocation of glucose transporter 4 (GLUT4) to the plasma membrane to facilitate cellular glucose uptake. Numerous observations indicate that the prime cause of type 2 diabetes mellitus (T2DM) is inflammation, the occurrence of which increases in obese individuals. Inflammatory mediators induce an insulin-resistance (IR) state where impaired insulin signalling fails to promote the glucose transporters for intracellular uptake of glucose. Hence compounds, which possess insulin-mimetic and anti-inflammatory potentials, may be effective in the treatment of obesity-induced IR during T2DM. Previous studies showed that vanadium oxo complexes possess insulin-mimetic activities whereas the tryptamine moiety offers anti-inflammatory potential. Hence a vanadyl-Schiff base complex (VOTP) consisting of the tryptamine moiety was synthesized by condensation of pyridoxal hydrochloride and tryptamine and its subsequent complexation with VOSO₄. HEK-293 cells, expressing a GLUT4-myc-GFP fusion protein, were treated with VOTP and GLUT4 translocation was quantified by total internal reflection fluorescence (TIRF) microscopy. Results indicated that VOTP could efficiently act as an insulin-mimetic substance. A high-content cell based assay using quantum dot-antibody conjugates showed that VOTP restored insulin signaling during IR by the inactivation of c-Jun N-terminal kinase-1 (JNK-1) and subsequent phosphorylation and activation of the tyrosine moiety of insulin receptor substrate (IRS). Also, high levels of phosphorylated Forkhead box O1 (FOXO) indicated low levels of gluconeogenesis. Hence VOTP has insulin-mimetic and anti-inflammatory potentials. Moreover, VOTP is highly effective at nanomolar treatment ranges, thus evades the toxicity issues. Collectively, these findings encourage us for future use of this compound as a potential anti-diabetic agent.

Full text of DOI:10.1039/c6ra11111d

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Page 1 of 30
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DOI: 10.1039/C6RA11111D
Insulinꢀmimetic and Antiꢀinflammatory Potential of a VanadylꢀSchiff Base Complex for its
Application against Diabetes
Jieun Ki†, Abhishek Mukherjee†, Sabarinathan Rangasamy†, Baskaran Purushothaman and
Joon Myong Song*
College of Pharmacy, Seoul National University, Seoul 151ꢀ742, South Korea
* Corresponding author
Prof. Joon Myong Song,
College of Pharmacy, Seoul National University, Seoul 151ꢀ742, South Korea
Tel: +82 2 880 7841; Fax: + 82 2 871 2238
Eꢀmail: jmsong@snu.ac.kr (Joon Myong Song)
†These authors contributed equally to this work.
1

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Abstract
Insulin signalling causes the translocation of glucose transporter 4 (GLUT4) to the plasma
membrane to facilitate cellular glucose uptake. Numerous observations indicate that the prime
cause of type 2 diabetes mellitus (T2DM) is inflammation, the occurrence of which increases in
obese individuals. Inflammatory mediators induce an insulinꢀresistance (IR) state where
impaired insulin signalling fails to promote the glucose transporters for intracellular uptake of
glucose. Hence compounds, which possess insulinꢀmimetic and antiꢀinflammatory potentials,
may be effective in the treatment of obesityꢀinduced IR during T2DM. Previous studies showed
that vanadium oxo complexes possess insulinꢀmimetic activities whereas tryptamine moiety
offers antiꢀinflammatory potential. Hence a vanadylꢀSchiff base complex (VOTP) consisting of
the tryptamine moiety was synthesized by condensation of pyridoxal hydrochloride and
tryptamine and its subsequent complexation with VOSO4. HEKꢀ293 cells, expressing a GLUT4ꢀ
mycꢀGFP fusion protein, were treated with VOTP and GLUT4 translocation was quantified by
total internal reflection fluorescence (TIRF) microscopy. Results indicated that VOTP could
efficiently act as an insulinꢀmimetic substance. Highꢀcontent cell based assay using quantum dotꢀ
antibody conjugates showed that VOTP restored insulin signaling during IR by the inactivation
of cꢀJun Nꢀterminal kinaseꢀ1 (JNKꢀ1) and subsequent phosphorylation and activation of tyrosine
moiety of insulin receptor substrate (IRS). Also, high levels of phosphorylated Forkhead box O1
(FOXO) indicated low levels of gluconeogenesis. Hence VOTP has insulinꢀmimetic and antiꢀ
inflammatory potentials. Moreover, VOTP is highly effective at nanomolar treatment ranges,
thus evades the toxicity issues. Collectively, these findings encourage us for future use of this
compound as a potential antiꢀdiabetic agent.
2

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Keywords: Insulin resistance; glucose transporter 4; total internal reflection fluorescence;
inflammation; bifunctional vanadium (IV) complex.
Abbreviation list
GLUT4: Glucose transporter 4; T2DM: Type 2 diabetes mellitus; IR: Insulin resistance; TIRF:
Total internal reflection fluorescence; JNK: cꢀJun Nꢀterminal kinaseꢀ1; IRS: Insulin receptor
substrate; FOXO: Forkhead box O1; TNFꢀα: Tumor necrosis factor alpha; DMEM: Dulbecco’s
modified Eagle’s medium; 2ꢀNBDG: 2ꢀ[Nꢀ(7ꢀnitrobenzꢀ2ꢀoxaꢀ1, 3ꢀdiazolꢀ4ꢀyl)amino]ꢀ2ꢀ deoxyꢀ
Dꢀglucose; GSK: Glycogen synthase kinase; PI3K: Phosphatidylinositolꢀ3ꢀkinase; IRSTK: IRS
tyrosine kinase.
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Introduction
The inability of cells to respond to insulin signalling is defined as insulin resistance (IR),
which constitutes the characteristic feature of type 2 diabetes mellitus (T2DM). The
chronic inflammatory response plays the key role in reduced insulin sensitivity. Patients
suffering from T2DM are often diagnosed with high levels of tumor necrosis factor alpha
(TNFꢀα). Recent reports implicated TNFꢀα as the major proꢀinflammatory cytokine
responsible for the pathogenesis of insulin resistance.1 Insulinꢀstimulated glucose disposal
is markedly reduced with increased expression of TNFꢀα.2 TNFꢀα inhibits the
phosphorylation of insulin receptor tyrosine by impeding tyrosine kinase of IRSꢀ1, thus
inhibiting insulin signalling.3 General therapeutic approaches include antiꢀinflammationꢀ
based combination to overcome inflammation and reduced sensitivity to insulin.
Accordingly, a compound possessing both antiꢀinflammatory and insulin mimetic
properties could contribute greatly to the achievement of efficient antidiabetic therapies.
A single compound with bifunctional therapeutic activity is very advantageous for
reducing the entire dose and improving the ease of drug formulation.
Regulation of glucose transporters (GLUT) constitutes another mode of TNFꢀα ꢀinduced
insulin resistance.4 GLUT proteins are glucose carriers that, upon insulin signalling,
mediate the facilitated diffusion of glucose down the concentration gradient inside of
cells. Most of those transporters reside on the cell membrane. However, GLUT4 is
located in intracellular vesicles in the basal state, either sequestered or inactive, and is
translocated to the plasma membrane upon insulin signalling to facilitate glucose uptake
by cells.5 TNFꢀα is found to be associated with low GLUTꢀ4 expression levels and
4

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inhibits insulinꢀstimulated GLUT4 translocation.6, 7 Impaired GLUT4 translocation is
believed to be the major cause of insulin resistance.
Vanadium oxo (VO) complexes, with improved solubility and reduced cytotoxicity, are
promising agents for the treatment of T2DM, as they possess insulinꢀmimetic properties.
The mechanism involves enhanced tyrosine phosphorylation in IRS and enhancement in
3ꢀ
insulinꢀstimulated GLUT4 translocation.8 Vanadyl (VO2+) is preferred to vanadate (VO4
) for use as an insulinꢀmimetic agent, as the former is less toxic to living cells.
The assessment of GLUT4 translocation poses a major challenge as GLUT4 cannot be
detected directly. Indirect methods, such as Western blot, ELISA, and immunoelectron
microscopic techniques are nonꢀsensitive, timeꢀconsuming, and cannot produce
quantitative data.9 Alternatively, different epitope tags and genetically encoded
fluorophores have been attached with GLUT4 for investigation of its cellular localization
and translocation.10 Evanescent field microscopy, commonly known as total internal
reflection fluorescence (TIRF) microscopy, constitutes an effective technique to
selectively excite and visualize fluorophores, as the evanescent angle restricts the imaging
of intracellular regions. TIRF allows imaging only near the plasma membrane and
minimizes background fluorescence, thus providing images with a higher signalꢀtoꢀnoise
ratio in comparison to those obtained in conventional epifluorescence studies. The
technique is efficient in quantifying the extent of protein translocation.
Until now, reports are scanty regarding a complex with insulinꢀmimetic and antiꢀ
inflammatory roles together, for its potential use during the IR state. Such a complex with
bifunctional activities could be a suitable alternative due to its low dosage frequency in
comparison to combination therapy. Here, we report a novel bifunctional vanadylꢀSchiff
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base complex (VOTP). TIRF was applied to quantify the translocation of GLUT4 to the
plasma membrane during an inflammationꢀinduced IR state by TNFꢀα treatment in HEKꢀ
293cells (Fig. 1A). The synthesized VOTP was applied to the inflammationꢀinduced IR
cellꢀbased assay using TIRF in order to assess its bifunctional potential to act as an antiꢀ
inflammatory agent and insulinꢀmimetic molecule in maintaining normal glucose
homeostasis during TNFꢀα induced inflammation and IR.
Materials and Methods
Plasmid Construct and Chemicals
The pBꢀGLUT4myc7ꢀGFP construct was purchased from Addgene (MA, USA). Human
insulin, wortmannin, glucose assay kit, and TNFꢀα were purchased from SigmaꢀAldrich
(St. Louis, MO, USA). Vanadium (IV) oxide sulfate hydrate was purchased from Sigmaꢀ
Aldrich Korea. Pyridoxal hydrochloride, Sodium acetate and tryptamine were purchased
from AlfaꢀAesar. All solvents were purchased from SigmaꢀAldrich Korea.
Synthesis of Schiff base Ligand (TPL)
TPL (Fig 1B (i)) was synthesized according to a previously described method.11 Pyridoxal
hydrochloride (0.5 mM) and tryptamine (0.5 mM) were dissolved in dry ethanol, stirred
and refluxed for 3 h under N2 atmosphere, and then the solvent was removed under
reduced pressure using a rotary evaporator. The product was precipitated by the addition
of acetonitrile. The precipitate was collected and dried under vacuum. Yield: 50%. Anal.
Calcd for C18H19N3O2: C, 69.88; H, 6.19; N, 13.58; O, 10.34; Found: C, 69.34; H, 6.56;
N, 13.73; O, 10.28. Selected IR peaks with tentative assignments (νmax, cm–1): 3457 (O–
H), 2963 (C–H), 1635 (C═N Schiff base coupled to the aromatic ring), 1011 (C–O
6

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1
phenol). FABꢀMS: 310 [M+1]. H NMR (DMSOꢀd6, δ/ppm): 2.37 (3H, s, CH3), 4.75
(4H, s, CH2 attached to OH), 7.24 (1H, s, CH), 7.26 (1H, s, CH), 7.95 (1H, s, CH=N),
10.4 (1H, s, NH), 13C NMR (500 MHz, DMSOꢀd6, δ/ppm): 136.26, 126.77, 123.22,
121.11, 118.96, 118.41, 118.04, 111.49, 58.48, 23.05, 19.62, 17.94.
Synthesis of bis(pyridoxylidenetryptamine) vanadium(IV) complex (VOTP)
VOTP was prepared by refluxing a mixture containing 0.5 mM of TPL and 0.25 mM of
VOSO4 in dry methanol for 15 h. Briefly 0.25 mM of tryptamine and 0.25 mM of
pyridoxal hydrochloride were taken in a roundꢀbottom flask, and 10 ml of dry methanol
was added. The reaction mixture was refluxed at 70⁰C for 3 h. The Schiff base ligand
formation (TPL) was confirmed by using TLC. 0.25 mM of VOSO4 was then dissolved in
5 ml of hot methanolic solution, and then added dropwise to the above ligand solution.
Finally, sodium acetate (1mM) was added. Heating continued for 12 h under N2
atmosphere. The excess solvent was removed using a rotary evaporator. The green color
solid (VOTP complex) was isolated and washed with diethyl ether and hexane. The
compound was dried under a vacuum condition and stored at 4⁰C. Yield: 50%. Anal.
Calcd for C36H36N6O5V: C, 63.25; H, 5.31; N, 12.29; Found: C, 63.34; H, 5.56; N, 12.73.
FAB mass: m/z 341. Selected IR peaks with tentative assignments (νmax, cm–1): 3457 (O–
H), 2963 (C–H), 1635 (C═N Schiff base coupled to the aromatic ring), 1011 (C–O
phenol), 978(V=O).
Cell culture and transfection
3T3ꢀL1 and HEKꢀ293 cells were obtained from the Korean Cell Line Bank (Seoul, South
Korea) and cultured in Dulbecco's modified Eagle's medium (DMEM) , supplemented
with 10% fetal bovine serum, 100 ꢁg/ml penicillin and 100 ꢁg/ml streptomycin at 37 °C
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in a humidified atmosphere of 5% CO2. HEKꢀ293 cells were kept overnight in serumꢀ
free DMEM without antibiotics, and transfected at 50ꢀ70% confluence with 1ꢀ5 ꢁg of pBꢀ
GLUT4myc7ꢀGFP plasmid using lipofectamineꢀ3000 (Invitrogen, CA, USA). Cells were
grown for 48 h, and the medium was replaced by DMEM containing FBS and antibiotics.
GLUT4ꢀGFP expression was monitored by a fluorescence microscope.
Glucose uptake assay
3T3ꢀL1 cells were seeded in 12ꢀwell cell culture plates (1×106 cells per well) and grown
overnight. Cells were then differentiated into adipocytes.12 The adipocytes were then kept
in serumꢀfree DMEM without antibiotics for 4 h, and treated with 100 nM of insulin or
different concentrations of VOTP (10 nM, 50 nM, 100 nM, and 200 nM) at 37 °C for 15
min. Each well was washed twice with Krebs Ringer Phosphate buffer (KRP buffer).
Then, cells were incubated with 100 µM of 2ꢀ[Nꢀ(7ꢀnitrobenzꢀ2ꢀoxaꢀ1, 3ꢀdiazolꢀ4ꢀ
yl)amino]ꢀ2ꢀ deoxyꢀDꢀglucose (2ꢀNBDG) at 37 °C for 10 min. After removal of the
incubation medium, the cells were washed three times with iceꢀcold PBS. The glucose
uptake by the cells was detected by measuring the fluorescent intensity of 2ꢀNBDG.
Images were acquired by a fluorescence microscope (IX73, Olympus, Tokyo, Japan)
using 488 nm excitation wavelength and 525 nm emission wavelength. Images were
processed using MetaMorph software, Version 7.1.3.0.
TIRF imaging studies
HEKꢀ293 cells were grown on coverslips in 6ꢀwell cell culture plates overnight. Cells
were then transfected with 1ꢀ5 ꢁg of pBꢀGLUT4myc7ꢀGFP plasmid using lipofectamineꢀ
3000 (Invitrogen, CA, USA) and allowed to grow for 48 h. Cells were pretreated with
VOTP or aspirin for 30 min in growth medium prior to TNFꢀα treatment and were treated
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with 10 ng/ml of TNFꢀα for 5 h. Insulin was treated for 30 min after TNFꢀα treatment.
Coverslips were then taken off and used for imaging of GFP fluorescence by TIRF
microscope (lgꢀTIRFM, TIRF Labs, NC, USA). All the images were taken at 20X
magnification and image analysis was performed using MetaMorph software, Version
7.1.3.0.
QdotꢀAb conjugation and imaging
The Abs against phospho forms of GSKꢀ3β, JNK, IRSser, FOXO, and IRStyr were
conjugated to Qdot525, QDot565, Qdot625, Qdot655, and Qdot705, respectively,
according to Tak et al.13 HepG2 cells were fixed in 4% formaldehyde, washed with PBS,
and treated with 0.2% saponin followed by washing. The cells were incubated with the
diluted QdotꢀAb conjugates at room temperature for 1 h. The cells were then washed with
PBS, and Qdot emission was detected using an Acusto optic tunable filter (AOTF)
(AOTF, TEAF10ꢀ0.45ꢀ0.7ꢀS, Brimrose Corporation, MD, USA) and a fluorescence
microscope (IX71, Olympus, Tokyo, Japan). All the images were taken at 20X
magnification and imaging analysis was performed using MetaMorph software, Version
7.1.3.0.
Statistical analysis
Each experiment was repeated three times. The statistical analysis was performed by oneꢀway
analysis of variance (ANOVA) to compare the means of two or more treatment groups, followed
by postꢀHoc multiple comparisons by the Tukey method. The difference was considered as
significant when p < 0.05.
Results and discussion
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Type II diabetes mellitus is a chronic disease, the rate of which increased dramatically during the
last two decades. The medicine currently used in the treatment of T2DM may cause a number of
detrimental side effects. On the other hand, insulin treatment is expensive, and regular injection
strategies are frequently painful to patients. Hence, it is important to screen for insulin mimetic
substances for effective treatment strategies against T2DM. Recent studies have identified a
strong correlation among obesity, inflammation, and T2DM. Inflammatory mediators like TNFꢀα
play significant role in the onset and progression of T2DM. Available drugs and phytochemicals
show promising insulin mimetic action. However, they are administered in high doses that cause
other undesirable symptoms. A molecule possessing insulin mimetic activity along with antiꢀ
inflammatory action activities may constitute a suitable alternative in treating T2DM. Earlier
studies have reported that vanadium oxo complexes possess insulinꢀmimetic activities.
Vanadium metal salts and their complexes are well studied in catalytic and biological
applications, such as antimicrobial, antitumor, and antiꢀleukaemia therapy.14,15 Vanadium can
form complexes with Schiff base ligands in different oxidation states to form compounds
possessing high stability and serve important biological applications as insulin mimetic agents.16
Schiff base ligands are called “privileged ligands”, and they are easily prepared by condensation
between an aldehyde and primary amine. We envisaged that choosing pydridoxal hydrochloride
and tryptamine would contribute greatly in enhancing antidiabetic and antiꢀinflammatory
properties. Pyridoxal 5′ꢀphosphate (PLP) and pyridoxamine (PM), the active forms of vitaminꢀ
B6, are known to inhibit the formation of advanced glycation endꢀproducts (AGEs), and hence
have been explored as active pharmaceutical agents for the treatment of diabetes.17 Recent
studies demonstrated that PLP can restrict the progression of diabetic nephropathy, and is
considered to constitute a promising candidate for the treatment of ageꢀrelated diabetic
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complications.18 Similarly, tryptamineꢀbased compounds were studied for antiꢀinflammatory
activities. Thus, it may be likely that utilizing pyridoxal hydrochloride and tryptamine in forming
a Schiff base ligand can produce a bifunctional activity along with vanadium.
Synthesis of Vanadium (IV) complex of pyridoxylidenetryptamine Schiff base complex
(VOTP)
Scheme (Fig. 1B (ii)) shows the oneꢀpot synthetic method utilized in the current study for the
preparation of vanadyl Schiff base complex (VOTP). A complexation reaction was performed
by the addition of vanadyl metal salt (VO2+) to the pyridoxal hydrochloride and tryptamine in the
presence of sodium acetate and the reaction mixture was refluxed for 12 h. The green powder
was isolated and washed with diethyl ether to obtain a vanadium (IV) complex of
pyridoxylidenetryptamine Schiff base (VOTP) in 50% yields.
Structural and spectral characterization of TPL and VOTP
The synthesized pyridoxylidenetryptamine Schiff base ligand (TPL) and VOTP were fully
characterized by FAB mass, FTꢀIR, electronic absorption spectroscopy, 1H NMR, and
TGA/DSC. The FAB mass spectrum of TPL showed a molecular ion peak [M+1] at m/z 310 that
revealed the formation of Schiff base ligand (supporting information Fig. S1). IR spectrum
analysis was performed in the range of 4000ꢀ400 cmꢀ1. The azomethine –C=N band appeared at
1495 cmꢀ1 in the TPL ligand (supporting information Fig. S3). This confirms the formation of an
imine bond during the condensation reaction of pydridoxal hydrochloride and tryptamine.
Further validation was achieved by 1H NMR spectrum, which showed a peak at δ=7.95 ppm for
–CH=N in the TPL ligand (supporting information Fig. S4). Similar characterization analyses
were conducted for the VOTP complex. The complex formation was verified by FAB mass
analysis. The FAB mass spectrum of charged species of VOTP showed a peak at m/z 341
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(supporting information Fig. S2). IR analysis of the VOTP complex showed a band at 1622 cmꢀ1
for azomethine –C=N and stretching vibration of the V=O bond at 978 cmꢀ1, indicating
coordination of vanadyl ion to the Schiff base. However, due to the paramagnetic (d1 system)
1
nature of vanadyl (IV), it was not feasible to record the H NMR spectrum for the VOTP
complex.19ꢀ20
Electronic absorption spectrum of TPL and VOTP
Figure 2 shows the electronic absorption spectra for the Schiff base ligand and its complex. The
spectra were measured in DMSO. The electronic spectra of TPL and VOTP exhibited a band at
284 nm, typical of nꢀπ* transition. It also revealed bands at 348 nm and 365 nm for ligand and
complex, respectively, which may be attributable to the ligandꢀbased πꢀπ* transition. The πꢀπ*
transition of VOTP showed a red shift of 17 nm compared with that of the ligand. This suggests
the coordination of metal ion to the ligand.
Thermal analysis
Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were carried out
to determine the thermal stability of the VOTP complexes. The TGA/DSC measurement for the
VOTP complex was performed in the range of ambient temperature to 800⁰C at a heating rate of
10⁰C per min. The obtained TGA/DSC curves are shown in Fig. 3. The TGA profile exhibited
three major distinctive decomposition steps. The first decomposition was observed between
room temperature and 120⁰C, which corresponded to a loss of water molecules in the VOTP
complex. The second decomposition step was observed around 253⁰C, which may be due to the
loss of the whole complex molecule. The third decomposition step was observed at 300 to
450⁰C, which may have arisen from the phase transformation of TPL ligand moiety. The total
mass loss value was 45.35%. The DSC curve showed one major peak and six small peaks. Below
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200⁰C, three thermal decomposition curves were observed, respectively, at 50⁰C, 100⁰C, and
150⁰C, which corresponded to the loss of external water molecules. The peak around 253⁰C
might be due to the decomposition of the VOTP complex. The broad DSC peak between 300 and
450◦C corresponded to calcination of the organic ligand TPL.
Insulin mimetic action of VOTP
We aimed at studying the GLUT4 translocation to the plasma membrane, which may be
considered as a marker to assess the insulin mimetic potential of compounds. The traditional wet
lab methods for the analysis of insulinꢀmimetic drugs suffer from the disadvantage of nonꢀ
specific signal intensities. Confocal microscopic analysis of fluorescent GLUT4 fusion proteins
or the Qꢀdot approach is insufficient to quantify the exact levels of GLUT4 translocation due to
the high noise generated from intracellular signalling. TIRF is a relatively new approach in
studying GLUT4 translocation. The fluorescent intensities detected at the evanescent field
specifically correspond to the membraneꢀassociated molecules without any intracellular noise
detection. As GLUT4 translocation is the major issue in addressing insulin signalling and T2DM,
effective visualization and quantitative analysis of localization results may be an effective way to
screen for antiꢀdiabetic and antiꢀinflammatory drugs. For optimization of VOTP concentration
for further experiments, the cellular glucose uptake was determined by measuring intracellular
glucose content in 3T3ꢀL1 adipocytes treated with different concentrations of VOTP. The
maximum glucose uptake was observed at 100 nM concentration of VOTP (Fig 4A v). Usually,
differentiated adipocytes or myotubes are used to study GLUT4 translocation. In this study, to
monitor the GLUT4 translocation we used HEKꢀ293 cells which are also widely used to study
the translocation of GLUTꢀ4 from the cytoplasm to the plasma membrane. Previous reports
revealed that HEKꢀ293 cells were effectively translocating the GLUTꢀ4 to the membrane by
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adopting similar signalling cascades observed in adipocytes and myotubes.21ꢀ23 The pBꢀ
GLUT4myc7ꢀGFP construct was transfected into HEKꢀ293 cells. The retroviral vector pB
contained a human GLUT4 cDNA, and cꢀmyc epitope tag fused with the green fluorescent
protein (GFP) coding sequence. A stable expression level of the GLUT4 reporter protein by the
transfected cells was monitored by the detection of fluorescence emitted by GFP. The
transfection efficiency was found to be almost 76%. The result for glucose uptake was further
confirmed by monitoring GLUT4 translocation by TIRF with different dosage treatment of
VOTP on HEKꢀ293 cells (Fig 4B). VOTP showed the potential of insulin mimetic activities even
at nanomolar treatment ranges. We optimized the dosage (100 nM VOTP) (Fig 4B iv) for further
studies in the IR model.
Antiꢀinflammatory potential of VOTP
In order to elucidate the role of VOTP during inflammationꢀinduced IR, we selected the TNFꢀα
induced IR model of HEKꢀ293 cells. Inflammation –induced IR state was already well
established methodology to study the glucose uptake and other metabolism associated with
glucose, which was well documented in adipocyte tissues and other cell types majorly
contributing the glucose metabolism such as HepGꢀ2 cells.24,25 TNFꢀα –induced IR state was also
26,27
previously reported in HEKꢀ293 cells.
The transfected cells were subjected to starvation
conditions before treatment with TNFꢀα or stimulation with insulin and/or VOTP. To confirm
the antiꢀinflammatory potential of VOTP, we compared the fluorescent signal intensities of
GLUT4ꢀGFP, as a measure of GLUT4 translocation to the plasma membrane, obtained by TIRF
microscopy from different experimental groups. Cells treated with TNFꢀα completely blocked
GLUT4 translocation. (Fig 5A i). Whereas, VOTP treatment along with the induction of IR by
TNFꢀα; significantly induced GLUT4 translocation. In addition, a marked increase in the level of
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GLUT4 translocation with respect to increase in VOTP dosage was also observed. (Fig 5A iiꢀv).
In another set of experiments, the cells were pretreated with VOTP in order to assess its antiꢀ
inflammatory potential during TNFꢀα induced IR. TIRF images showed that TNFꢀα induced IR
state completely inhibited the insulin signalling (Fig 3B i). Interestingly, the preꢀtreatment of
VOTP prior to IR induction effectively restored the insulin signalling in TNFꢀα treated HEKꢀ
293 cells, the extent of which increased with increase in VOTP concentration and attained the
maximum level at 100 nM of VOTP pretreatment (Fig 5B iiꢀv).Equal dosage of VOTP and
insulin, induced a comparable extent of GLUT4 translocation (Fig 6A).
To compare the antiꢀinflammatory potential of VOTP, we used aspirin, a known antiꢀ
inflammatory drug, during IR. TIRF results showed that VOTP (100 nM) preꢀtreatment could
induce significant extent of GLUT4 translocation in absence of insulin even after the induction
of IR (Fig 6B iii). we also observed that, VOTP (100 nM) preꢀtreatment exhibited much better
ability in restoring insulin signalling and GLUT4 translocation (Fig 6B v), even at much lower
concentrations in comparison to aspirin treatment (100 µM) (Fig 6B iv). The mode of activation
of the Insulin mimetic drugs is independent of IRSꢀtyrosine kinase (IRSTK). Instead, they are
processed via activation of phosphatidylinositolꢀ3ꢀkinase (PI3K) which, in turn, activates IRS by
phosphorylation of its tyrosine moiety. We applied wortmannin, a potent inhibitor of PI3K, to
assess the role of the kinase in VOTP induced IRS activation. Wortmannin pretreatment
decreased the extent of GLUT4 translocation; however it could not completely block the GLUT4
translocation of VOTPꢀpretreated cells prior to IR induction (Fig 6B vi). In general, PI3K
inhibition hampers the action of insulin mimetic drugs. Accordingly, the insulin mimetic action
of VOTP should be unable to induce GLUT4 translocation and intracellular glucose uptake.
Hence, we explored that VOTP may act via a different mechanism to stimulate GLUT4
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translocation and to restore insulin signalling in our experimental model. The most evident
mechanism may be its antiꢀinflammatory action, which triggers the reactivation of IRSTK to
phosphorylate the IRS tyrosine moiety. This idea was obviously supported by the data of
wortmanninꢀtreated cells, in which VOTP treatment induced significant GLUT4 translocation
(Fig 6B vi).
To further confirm the antiꢀinflammatory potential of VOTP, the activity levels of certain
intracellular kinases, which are related to inflammationꢀinduced IR were determined by highꢀ
content monitoring using QDot antibody (Ab) conjugates. Highꢀcontent monitoring does not
require cell lysis, and several biomolecules can be quantified concurrently from a single sample
in a single run. High fluorescent intensity of QDots leads to high resolution images and accuracy
in quantification. Generally, TNFꢀα induces IR via the phosphorylation and activation of JNKꢀ1.
It also triggers the inactivation of glycogen synthase kinase (GSK), decreases the level of
phosphorylated FOXO1, and inhibits the phosphorylation and subsequent activation of IRS
tyrosine (IRStyr).28 In T2DM, adipocytes are critically involved in the establishment of
inflammatory state of the system by secreting various inflammatory cytokines including TNFꢀα
in association with tissue infiltrating macrophages. However, in liver and muscle cells the
glucose uptake was reported to be impaired due to the insulin resistance established by
inflammatory state. 29, 30 Hence, we used HepG2 cells to study the antiꢀinflammatory potential of
VOTP and its role in regulating the intracellular kinases associated with inflammation induced
IR state. HepG2 cells are a liver cell (Meijer et al., 2015). HepG2 cells pretreated with VOTP
followed by IR induction significantly reduced the level of phosphorylated JNKꢀ1 with a
concomitant increase in the levels of phosphorylated forms of IRStyr and GSK3β (Fig 7A iv). In
addition, VOTP produced a marked increase in the levels of phosphorylated FOXO, thus
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stopping gluconeogenesis, which otherwise depends on the level of nonꢀphosphorylated FOXO
(Fig 7A iv). The low levels of the phosphorylated form of IRS serine (IRSser) and high levels of
phosphorylated IRStyr (Fig 7A iv) confirmed that VOTP pretreatment caused the activation of
IRSTK, which is otherwise deactivated during inflammationꢀinduced IR. The applied VO
complex plays a bifunctional role in maintaining normal glucose homeostasis. Specifically, it
may: (i) act by activating IRSꢀtyrosine kinase during the inflamed condition, which reflects its
antiꢀinflammatory potential; or (ii) act by the PI3K dependent pathway, which reflects its insulin
mimetic potential. Inflammation plays a major role in the onset and progression of T2DM. In
T2DM, the activation of IRS is blocked by the inactivation of IRSTK that otherwise
phosphorylates and activates IRStyr for effective insulin signalling. Thus, an effective way to
treat T2DM is to utilize antiꢀinflammatory drugs to restore insulin signalling. Moreover, insulinꢀ
mimetic drugs are also applied to maintain normal glucose homeostasis. The synthesized vanadyl
complex possesses both of these properties and can constitute a suitable alternative to traditional
drugs. The compound is effective at the nanomolar treatment range that is generally nonꢀtoxic to
living cells. The screening system to quantify the GLUT4 translocation involving a combination
of an inflammationꢀinduced IR cellular model and TIRF demonstrates effectiveness in analysing
such compounds in a convenient manner with high precision.
Conclusions
The current study constitutes perhaps the pioneering report of a synthesized vanadyl
complex possessing insulinꢀmimetic and antiꢀinflammatory properties together. As the
complex is highly active at very low doses, it may find a suitable role as an alternative
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over traditional drugs for treating T2DM. Further research is necessary in order to assess
the adverse effects of this complex, if any, in animal models.
Acknowledgements
We are grateful to the Research Institute of Pharmaceutical Sciences at Seoul National
University for providing experimental equipment. This work was supported by National
Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and
Technology (MEST) (2015R1A2A1A05001842).
Declarations of interest
The authors declare no competing financial interests.
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Figure captions
Figure 1. Schematic representation: (A) Insulin signaling pathway and GLUT4 translocation.
Binding of insulin to its receptor induces phosphorylation, followed by the recruitment of IRS,
which ultimately leads to the translocation of GLUT4ꢀcontaining vesicles from cytosol to the
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plasma membrane. TNFꢀα blocks the phosphorylation and activation of insulin receptor to
deactivate the downstream kinases and blocks GLUT4 translocation and glucose uptake by
cells. VOTP plays an antiꢀinflammatory role against TNFꢀα induced insulin resistance. Cells
expressing GLUT4ꢀmycꢀGFP were grown on coverslip glass. Fluorophores localized at the
evanescent field (plasma membrane) only, could be visualized by TIRF microscopy. (B) (i)
Chemical structure of the Schiff base ligand (TPL) and (ii) Oneꢀpot synthesis of bifunctional
(pyridoxylidinetryptamine) vanadium (IV) complex (VOTP).
Figure 2. UVꢀVisible Absorption spectra of TPL and VOTP.
Figure 3. TGA/DSC profile curves of VOTP complex.
Figure 4. (A) Effect of different concentrations of VOTP on glucose uptake by
differentiated 3T3ꢀL1 adipocytes. Insulin was used to compare the insulinꢀmimetic efficacy of
VOTP. (i) control; (ii) insulin (100 nm); (iii) VOTP (10 nm); (iv) VOTP (50 nm); (v) VOTP
(100 nm); (vi) VOTP (200 nm). (vii) shows the respective fluorescence intensities that
correspond to the amount of glucose uptake. (B) Quantification of VOTPꢀinduced GLUT4
translocation in HEKꢀ293 cells by using TIRF microscopy (i) control; (ii) VOTP (10 nm);
(iii) VOTP (50 nm); (iv) VOTP (100 nm); (v) VOTP (200 nm). (vi) Shows the respective
fluorescence intensities that correspond to the amount of glucose uptake in HEKꢀ293 cells in
response to the different concentration of VOTP. The data of insulin and VOTP treated groups
were compared with those of the control group. ***p <0.001 and **p <0.01 vs control.
Figure 5. (A) Effect of VOTP on GLUT4 translocation during TNFꢀα induced IR state in
the HEKꢀ293 cells. (i) Control;(ii) VOTP (10 nm); (iii) VOTP (50 nm); (iv) VOTP (100 nm);
(v) VOTP (200 nm). (vi) shows the respective fluorescence intensities that correspond to the
amount of GLUTꢀ4 translocation. The data of (TNFꢀα+VOTP) treated groups were compared
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with those of the TNFꢀα treated group. (B) Effect of pretreatment of VOTP on insulin
signalling as assessed by measuring GLUT4 translocation during TNFꢀα induced IR state.
(i) control; (ii) VOTP (10 nm); (iii) VOTP (50 nm); (iv) VOTP (100 nm); (v) VOTP (200 nm).
(vi) Shows the respective fluorescence intensities that correspond to the amount of glucose
uptake. The data of (VOTP+TNFꢀα) treated groups were compared with those of the TNFꢀα
treated group. *p<0.05; **p<0.01; ***p<0.001 vs control.
Figure 6. (A) Comparison of the insulinꢀmimetic potential of VOTP. (i) control; (ii) insulin
(100 nm); (iii) VOTP (100 nm). (iv) Shows the representative histogram corresponding to the
fluorescence intensities of GFP as a measure of GLUT4 translocation. The data of insulin and
VOTP treated groups were compared with those of the control group. (B) Assessment of the
antiꢀinflammatory potential of VOTP. (i) control; (ii) TNFꢀα+insulin; (iii) VOTP (100
nm)+TNFꢀα; (iv) aspirin (100 ꢁM)+TNFꢀα+insulin; (v) VOTP (100 nm)+TNFꢀα+insulin; (vi)
wortmannin+VOTP (100 nm)+TNFꢀα+insulin. Cells were treated with VOTP or aspirin prior to
TNFꢀα treatment. Insulin (100 nm) was applied after the TNFꢀα treatment. (vii) Shows the
representative histogram corresponding to the fluorescence intensities of GFP as a measure of
GLUT4 translocation. The data of the control group were compared with those of the (TNFꢀ
α+insulin) treated group. The data of TNFꢀα+VOTP/aspirin treated groups were compared with
those of the (TNFꢀα+insulin) treated group. *p<0.05; ***p<0.001 vs control
Figure 7. (A) Highꢀcontent multicolor cellular images showing simultaneous monitoring of
the upregulation/downregulation of insulin downstream signal molecules pꢀGSK3β, pꢀ
JNK1, pIRSser, pꢀFOXO1, and pꢀIRStyr. (i) control; (ii) insulin; (iii) aspirin (100 ꢁM)+TNFꢀ
α+insulin; (iv) VOTP (100 nm)+TNFꢀα+insulin. HepG2 cells were treated with VOTP or
aspirin prior to TNFꢀα treatment. Insulin was applied after the TNFꢀα treatment. The images
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(10X) were acquired using a fluorescent microscope (Model) equipped with AOTF (Company).
(B) Representative histogram corresponding to the fluorescence intensities of QDots. The
data of the control group were compared with those of the TNFꢀα+insulin treated group. The
data of TNFꢀα+VOTP/aspirin treated groups were compared with those of the TNFꢀα+insulin
treated group. **p<0.01; ***p<0.001 vs control.
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Figure 3
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