In vitro anti-hyperglycemic activity of 4-hydroxyisoleucine derivatives

Article abstract of DOI:10.1016/j.phymed.2014.09.007

The nonproteinogenic amino acid, 4-hydroxyisoleucine (1) has been isolated in large quantities from the fenugreek (T. foenum-graecum) seeds. Few novel derivatives (3-11 and 13-18) were prepared from the naturally occurring 4-hydroxyisoleucine (1) and scre

Full text of DOI:10.1016/j.phymed.2014.09.007

Phytomedicine 22 (2015) 66–70
Contents lists available at ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
In vitro anti-hyperglycemic activity of 4-hydroxyisoleucine derivatives
a
b
a
b
Venkateswarlu Korthikunta , Jyotsana Pandey , Rohit Singh , Rohit Srivastava ,
b
b,∗
a,∗
Arvind K. Srivastava , Akhilesh K. Tamrakar , Narender Tadigoppula
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, UP 226 031, India
a
b
Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, UP 226 031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 April 2014
Received in revised form 11 August 2014
Accepted 13 September 2014
The nonproteinogenic amino acid, 4-hydroxyisoleucine (1) has been isolated in large quantities from the
fenugreek (T. foenum-graecum) seeds. Few novel derivatives (3–11 and 13–18) were prepared from the
naturally occurring 4-hydroxyisoleucine (1) and screened for their in vitro glucose uptake stimulatory
effect in L-6 skeletal muscle cells. The derivatives 6, 7, 8, 10 and 11 exhibited better glucose uptake
stimulatory activity than parent compound, 4-hydroxyisoleucine at 5 and 10 ␮M concentrations and
compounds 7 and 11 enhanced translocation of insulin sensitive glucose transporters-4 in skeletal muscle
cells.
Keywords:
4
-Hydroxyisoleucine
Fenugreek seeds
Anti-hyperglycemic activity
Glucose uptake
©
2014 Elsevier GmbH. All rights reserved.
GLUT4 translocation
Introduction
like skeletal muscle and fat. Hence, interventions with ability to
stimulate glucose uptake might be important for the treatment
of diabetes mellitus. Although numerous oral anti-hyperglycemic
drugs exist along with insulin, there is no promising therapy for
type 2 diabetes mellitus.
Diabetes mellitus is a metabolic disorder characterized by
the presence of hyperglycemia due to defective insulin secre-
tion, defective insulin action or both (Mitra, 2008). The chronic
hyperglycemia of diabetes is associated with relatively specific
long-term microvascular complications affecting the eyes, kidneys
and nerves, as well as an increased risk for cardiovascular disease
Natural products have played a key role in the discovery of
antidiabetic drugs (Fig. 1) (Kanaujia et al., 2010) Metformin is
currently used as antidiabetic agent in the treatment of type 2
diabetes. Metformin and its analogues (Shapiro et al., 1959) were
synthesized on the basis of a natural product lead, that is, galegine
(Bailey and Day, 1989). Acarbose, a complex of oligosaccharides
isolated from Actinoplanes sp., was discovered by Bayer pharma-
ceuticals for a search of ␣-glucosidase inhibitors (Shu, 1998).
Acarbose is an antidiabetic agent to treat type 2 diabetes. Few
plant-derived unusual amino acids have been reported for their
anti-hyperglycemic activity. Hypoglycin A and B isolated from the
fruits of Blighia sapida, which are chemically related to lysine were
reported for their anti-hyperglycemic activity (Hassall and Reyle,
1954; Feng and Patrick, 1958). Enormous amount of research work
is going on 4-hydroxyisoleucine (4-HIL), due to its broad range of
pharmaceutical activities as insulinotropic, anti-dyslipidemic, and
anti-hyperglycemic agent (Sauvaire et al., 1998; Narender et al.,
2006)
(
CVD). It affects all age group worldwide. In 1985, an estimated 30
million people around the world were diagnosed with diabetes;
in 2000, that figure rose to over 150 million; and in 2012, the
International Diabetes Federation (IDF) estimated that 371 mil-
lion people had diabetes (International Diabetes Federation, 2012,
www.idf.org/diabetesatlas). That number is projected to rise up
to 552 million (or 1 in 10 adults) by 2030, which equates to 3
new cases per second (International Diabetes Federation, 2012,
www.idf.org/diabetesatlas).
The main pathophysiological feature of type 2 diabetes melli-
tus is insulin resistance, characterized by reduced ability of target
tissues, such as the liver, skeletal muscle, and adipose, to respond
to insulin (Reaven, 1995). This includes impairment in the insulin
stimulated translocation of GLUT4 to cell surface and resulting
defect in insulin-stimulated glucose uptake in peripheral tissues
The 4-hydroxyisoleucine was first isolated as free acid from
fenugreek (T. foenum-graecum) seeds (Fowden et al., 1973) and
structure was validated by X-ray crystallography (Alcock et al.,
∗ Corresponding authors. Tel.: +91 522 2772450.
E-mail addresses: akhilesh tamrakar@cdri.res.in (A.K. Tamrakar),
t narendra@cdri.res.in, tnarender@rediffmail.com (N. Tadigoppula).
1
989), establishing the absolute stereochemistry as 2S, 3R, 4S. Fur-
ther, the SAR studies indicated that the absolute configuration
http://dx.doi.org/10.1016/j.phymed.2014.09.007
0
944-7113/© 2014 Elsevier GmbH. All rights reserved.

V. Korthikunta et al. / Phytomedicine 22 (2015) 66–70
67
NH NH
Glucose uptake
NH
OH NH2
COOH
N
N
H
NH2
H2N
N
H
The determination of 2-DG uptake in L6-GLUT4myc myotubes
was performed as described previously (Tamrakar et al., 2010).
Briefly myotubes were incubated with test compounds for 16 h
under culture condition. Glucose uptake was assessed for 5 min
in HEPES-buffered saline [140 mM NaCl, 20 mM HEPES, 5 mM KCl,
Metformin
Galegine
4-Hydroxyisoleucine
OH
HO
HO
H3C
OH
OH
OH
OH
2.5 mM MgSO , 1 mM CaCl (pH 7.4)] containing 10 ␮M 2-DG
4 2
N
3
H
(0.5 ␮Ci/ml 2-[ H]-DG) at room temperature. Subsequently cells
were rinsed with ice-cold saline solution. To quantify the radioac-
tivity incorporated, cells were lysed with 0.05 N NaOH and lysates
were counted with scintillation fluid in a ␤-counter. Nonspecific
uptake was determined in the presence of cytochalasin B (50 ␮M)
during the assay, and these values were subtracted from all other
values. Glucose uptake measured in triplicate and normalized to
total protein, was expressed as percent induction with respect to
unstimulated cells.
OH
OH HO
OH
O
HO
OH
O
N
H
HO
O H OH
Acarbose
Fagomine
OH
O
HN
COOH
H N
COOH
2
HO
O
N
H
COOH
NH2
Hypoglycin B
GLUT4 translocation
Hypoglycin A
4- Hydroxypipeco lic acid
Fig. 1. Naturally occurring and synthetic antidiabetic agents.
GLUT4 level at the cell surface of non-permeabilized L6-
GLUT4myc myotubes was measured by an antibody-coupled
colorimetric assay as described previously (Tamrakar et al., 2011).
Briefly, after the indicated treatments, cells were washed in ice-cold
PBS (154 mM NaCl, 5.6 mM Na HPO , 1.1 mM KH PO ) supple-
of 2S, 3R, 4S stereogenic centres has considerable influence on
their pharmaceutical activity (Christophe et al., 2000; Broca et al.,
999). Recently we have reported the lipid lowering activity of 4-
hydroxyisoleucine in high fat diet fed hamster model (Narender
et al., 2006). We have also reported the glucose uptake stimulatory
effect of 4-HIL mediated by enhanced translocation of insulin sen-
sitive glucose transporters-4 in skeletal muscle cells (Jaiswal et al.,
012). Thus, 4-HIL seems a very promising dietary supplement in
the treatment and management of diabetes mellitus. Therefore,
we isolated a large quantity of 4-HIL (1) and prepared a series of
-hydroxyisoleucine derivatives (3–11 and 13–18) and evaluated
2
4
2
4
1
mented with 1 mM CaCl and 1 mM MgCl (pH 7.4). Cells were then
2
2
fixed in 3% paraformaldehyde for 30 min and quenched in 100 mM
◦
glycine for 10 min, all at 4 C. Cells were blocked in 5% skimmed
milk for 15 min and then incubated with anti-myc antibody solu-
◦
tion (1.0 ␮g/ml in PBS with 3% skimmed milk) for 1 h at 4 C. After
2
labelling, excess antibodies were removed by extensive washing
in ice-cold PBS. Cell surface GLUT4-bound antibodies were probed
by HRP-conjugated secondary antibodies followed by detection of
bound HRP by O-phenylenediamide assay. The fraction of GLUT4
at the cell surface, measured in triplicate, was expressed as fold
induction with respect to unstimulated cells.
4
their in vitro glucose uptake stimulatory effect in skeletal muscle
cells.
Materials and methods
Results and discussion
Isolation of 4-hydroxyisoleucine
Chemistry
The seeds (20 kg) of T. foenum-graecum were collected from the
local market of Lucknow and extracted with 16 l of ethyl alcohol
four times in a percolator. The resultant alcoholic extracts were
The synthesis of derivatives of pure 4-HIL (1) was accomplished
by subjecting the acetylation of 4-hydroxyisoleucine by using acetic
anhydride in pyridine to afford the N-acetylated lactone (2). The
(
4
64 l) combined and concentrated under reduced pressure to give
00 g of alcohol extract. This was fractionated with chloroform
◦
resultant compound 2 was treated with pyrrolidine in DCM at 0 C
for overnight to afford the 4-HIL amide (Nicolas et al., 2006) (3). Fur-
ther this amide 3 was treated with acetic anhydride in presence of
pyridine at room temperature to afford corresponding O-acetylated
derivative (4) (Scheme 1).
and n-butanol successively. The resultant aqueous fraction (40 g)
was subjected to conventional silica gel column chromatography
using ethyl acetate and methanol (90:10) solvent system to give
an unusual amino acid (2 g). It was characterized as 4 hydroxy-
1
13
Furthermore the synthesis of N-Boc protected lactone (5) was
isoleucine by using H NMR, C NMR, IR and mass spectral data
and comparing with literature data (Fowden et al., 1973; Alcock
et al., 1989).
accomplished by subjecting the 4-HIL to the anhydride Boc O in
2
the presence of base to obtain the compound 5. The resultant lac-
tone (5) was converted in to amides (Huang et al., 2001) by using
DIBAL–H–H NR complexes in THF or pyrrolidine in DCM system to
afford the compounds 6–11 in good yields (Scheme 2).
2
Cell culture
Furthermore N-tosylated amides of 4-HIL (14–18) were synthe-
sized from 4-HIL as shown in Scheme 3. Treatment of lactone (12),
L6 skeletal muscle cells stably expressing rat GLUT4 with a
myc epitope inserted in the first exofacial loop (L6-GLUT4myc),
a kind gift of Dr Amira Klip, Program Cell Biology, The Hospi-
tal for Sick Children, Toronto, Canada, were maintained in DMEM
supplemented with 10% FBS and 1% antibiotic/antimycotic solu-
tion (10,000 U/ml penicillin G, 10 mg/ml streptomycin, 25 ␮g/ml
amphotericin B) in a humidified atmosphere of air and 5% CO at
2
◦
3
7 C. Differentiation was induced by switching confluent cells to
medium supplemented with 2% FBS. Experiments were performed
in differentiated myotubes 6–7 days after seeding.
Scheme 1. Synthesis of 4-HIL derivatives.

6
8
V. Korthikunta et al. / Phytomedicine 22 (2015) 66–70
Scheme 2. Synthesis of N-Boc protected 4-HIL derivatives.
which was obtained from 4-HIL in the presence of 3 N HCl, with
◦
tosylchloride in dry DCM at 0 C to room temperature for overnight
afforded the N-tosylated lactone (Nicolas et al., 2006) (13). Then
the resultant lactone was subjected to aminolysis (Nicolas et al.,
2
006) by treating with DIBAL–H–H NR complex in THF to afford
2
the amides 14–18 in good yields.
Fig. 2. Dose dependent effect of 4-HIL (1) and its derivatives (7 and 11) on glu-
cose uptake in L6-GLUT4myc skeletal muscle cell lines. Results are expressed as fold
stimulation over control. *p < 0.05, ***p < 0.001 relative to control.
Biological evaluation Glucose uptake in L6 skeletal muscle cell
lines
compound showed poor to no effect on glucose uptake in L-6 mus-
cle cells at 10 ␮M concentration.
To check the in vitro antidiabetic effect, all the compounds were
evaluated for their effect on glucose uptake in L6-GLUT4myc skele-
tal muscle cell lines. Skeletal muscle is the major insulin-target
tissues responsible for maintenance of whole body glucose homeo-
stasis. Insulin stimulates cellular glucose uptake in muscle tissues
by inducing the translocation of glucose transporter-4 (GLUT4)
from an intracellular pool to the plasma membrane. In the dia-
betic state, insulin stimulated translocation of GLUT4 gets impaired
leading to decreased glucose uptake by muscle cells, which sig-
nificantly contribute to the elevated blood glucose levels. Cells
were differentiated to myotube stage and treated overnight with
indicated concentration of compounds under culture conditions
followed by determination of glucose uptake according to Hwang
et al. (2008) with some minor modifications. The results of the glu-
cose uptake stimulatory effect of compounds are summarized in
Table 1
It is further assessed whether the ability of 7 and 11 to stimu-
late glucose uptake was mediated by potentiation of insulin action
or activation of cellular processes independent of the hormone.
Cells were treated with 7 and 11 (10 ␮M) for 16 h with final three
hour in serum-deprived medium and a subset of cells was stim-
ulated with insulin (100 nM) for 20 min before the measurement
of glucose uptake. As shown in Fig. 3, insulin alone caused 2.2-
fold (p < 0.001 vs. control basal) stimulation of glucose uptake in
L6 myotubes over basal state. Pretreatment with 7 and 11 (16 h)
caused further increase in insulin signal, suggesting that the effect
of 7 or 11 and insulin on glucose transport is additive. But the gain
in transport was lower than that calculated from the independent
effect of compounds (7 and 11) and the insulin. These observations
suggest that compounds 7, 11 and insulin activate glucose transport
by different sub cellular pathways, but some elements common to
the action of both agents may be finite.
The naturally occurring compound 4-HIL (1) enhanced the glu-
cose uptake by 38.67% in L6 muscle cells at 10 ␮M concentration
Effect of 4-HIL derivatives on GLUT4 translocation to cell surface
in L-6 GLUT4myc skeletal muscle cell lines
(Table 1). From the tested compounds 3–11 and 13–18, compound 7
and 11 showed maximal stimulation of glucose uptake 109.5% (2.10
folds vs control) and 105.25% (2.05 folds vs control) respectively at
To check whether the increase in glucose uptake by the
compounds is attributable to the translocation of GLUT4 from
intercellular compartment to plasma membrane, surface level
1
0 ␮M concentration (Table 1). Further these two compounds were
evaluated for their dose-dependent effect using different concen-
trations of compounds (Fig. 2). In L6 cells compound 7 enhanced
the glucose uptake 70.5% (1.71 folds vs control basal) at 5 ␮M
concentration and 122.5% (2.22 folds vs control basal) at 25 ␮M con-
centrations where as compound 11 enhanced the glucose uptake
by 62.7% (1.63 folds vs control basal) and 143.0 (2.43 folds vs con-
trol basal) at 5 ␮M and 25 ␮M concentrations respectively (Table 1;
Fig. 2). From the remaining compounds, 6, 8 and 10 also signifi-
cantly increased the glucose uptake by 55.4% (1.55 folds vs control
basal), 66.0% (1.66 folds vs control basal) and 50.25% (1.55 folds
vs control basal) at 10 ␮M concentrations respectively (Table 1).
Compounds 6, 7, 8, 10 and 11 also exhibited better glucose uptake
activity than it’s naturally occurring 4-HIL (1). All other synthesized
Fig. 3. Effect of 4-HIL – derivatives (7 and 11) on insulin stimulated glucose uptake
in L6-GLUT4myc skeletal muscle cell lines. Results are expressed as fold stimulation
over control basal. *p < 0.05, ***p < 0.001 relative to control.
Scheme 3. Synthesis of N-Ts protected 4-HIL derivatives.

V. Korthikunta et al. / Phytomedicine 22 (2015) 66–70
69
Table 1
In vitro glucose uptake in L6-GLUT4myc skeletal muscle cells of 4-HIL derivatives.
R
1
O
OR HN
R
HN
1
O
O
R2
.
Compound
1
R
R1
R2
Concen-tration (␮M)
% Glucose uptake
16.0
No. folds vs basal
5
10
1.16
1.38
2.16
*
H
H
OH
38.67
*
**
25
116.0
3
H
10
32.50
1.33
4
5
6
10
10
10
10.50
9.00
1.11
1.09
1.55
–
–
H
55.4*
5
10
5
70.5*
1.71
2.10
2.22
*
**
7
8
H
H
109.50
*
**
2
122.0
10
66.00*
1.66
9
H
H
10
10
−12.00
0.88
1.50
1
0
50.25*
62.7*
5
10
1.63
2.05
2.43
*
**
1
1
1
3
H
–
105.25
*
**
25
143.0
–
10
10
10
−1.00
8.50
0.99
1.09
1.02
1
4
5
H
H
O
S
O
HN
1
2.23
OMe
OMe
H
O
S
O
N
16
17
18
H
H
H
10
10
−14.00
−7.50
0.86
0.93
H
N
O
S
O
OMe
OMe
H
N
O
S
O
10
10
10.50
96.5**
1.11
1.96
Cl
Rosiglitazone
*
p < 0.05.
p < 0.01.
p < 0.001 relative to control.
*
*
*
**
of GLUT4myc was measured by an antibody-coupled assay
Tamrakar et al., 2011). Prolonged exposure (16 h) of L6-GLUT4myc
myotubes to compounds 7 and 11 caused a substantial increase
concentration-dependent fashion (Fig. 4). A significant aug-
mentation in surface level of GLUT4 was observed at 10 ␮M
(p < 0.01) concentration, reaching to the maximal level at 25 ␮M
(p < 0.01). Insulin alone (100 nM), when added 20 min before
(
in the level of GLUT4 molecules at the plasma membrane in a

7
0
V. Korthikunta et al. / Phytomedicine 22 (2015) 66–70
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.phymed.
2
014.09.007.
References
Alcock, N.W., Crout, D.H.G., Gregorio, M.V.M., Lee, E., Pike, G., Samuel, C.J., 1989.
Stereochemistry of the 4-hydroxyisoleucine from Trigonella foenum-graecum.
Phytochemistry 28, 1835–1841.
Bailey, C.J., Day, C., 1989. Traditional plant medicines as treatments for diabetes.
Diabetic Care 12, 553–564.
Kanaujia, A., Duggar, R., Pannakal, S.T., Yadav, S.S., Katiyar, C.K., Bansal, V., Anand, S.,
Sujatha, S., Lakshmi, B.S., 2010. Insulinomimetic activity of two new gallotannins
from the fruits of Capparis moonii. Bioorg. Med. Chem. 18, 3940–3945.
Broca, C., Rene, G., Pierre, P., Yves, S., Manteghetti, M., Tournier, M., Masiello, P.,
Gomis, R., Ribes, G., 1999. 4-Hydroxyisoleucine: experimental evidence of its
insulinotropic and antidiabetic properties. Am. J. Physiol. 277, 617–623.
Christophe, B., Manteghetti, M., Gross, R., Baissac, Y., Jacob, M., Petit, P., Sauvaire, Y.,
Ribes, G., 2000. 4-Hydroxyisoleucine: effects of synthetic and natural analogues
on insulin secretion. Eur. J. Pharmacol. 390, 339–345.
Fig. 4. Effect of 4-HIL derivatives on GLUT4 translocation to cell surface in L-
GLUT4myc skeletal muscle cell lines. Results are expressed as fold stimulation
over control basal. *p < 0.05, **p < 0.01 relative to respective control condition,
6
#
##
p < 0.001 relative to control basal.
Feng, P.C., Patrick, S.J., 1958. Studies of the action of hypoglycin-A, an hypoglycaemic
substance. Br. J. Pharmacol. Chemother. 13, 123–130.
Fowden, L., Pratt, H.M., Smith, A., 1973. 4-Hydroxyisoleucine from seed of Trigonella
foenum-graecum. Phytochemistry 12, 1707–1711.
the measurement of GLUT4 translocation resulted in 2.2-fold
p < 0.001) increase in surface level of GLUT4 compared to basal
state in L6-GLUT4myc (Fig. 4). Treatment with 7 and 11 caused
significant enhancement of insulin stimulated translocation of
GLUT4myc to cell surface (p < 0.01) compared to control insulin
treated (Fig. 4).
(
Hassall, C.H., Reyle, K., 1954. Hypoglycin A, B: biologically active polypeptides from
Blighia sapida. Nature 173, 356–357.
1
2
Huang, P., Zheng, X., Deng, X.M., 2001. DIBAL–H–H2NR and DIBAL–H–HNR R ·HCl
complexes for efficient conversion of lactones and esters to amides. Tetrahedron
Lett. 42, 9039–9041.
Hwang, S.L., Yang, B.K., Lee, J.Y., Kim, J.H., Kim, B.D., Suh, K.H., Kim, D.Y., Kim, M.S.,
Song, H., Park, B.S., Huh, T.L., 2008. Isodihydrocapsiate stimulates plasma glucose
uptake by activation of AMP-activated protein kinase. Biochem. Biophys. Res.
Commun. 371, 289–293.
Jaiswal, N., Maurya, C.K., Venkateswarlu, K., Sukanya, P., Srivastava, A.K., Naren-
der, T., Tamrakar, A.K., 2012. 4-Hydroxyisoleucine stimulates glucose uptake by
increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-
Conclusion
In conclusion, we isolated the nonproteinogenic amino acid (2S,
3
R, 4S)-4-hydroxyisoleucine from seeds of fenugreek in large quan-
tity and prepared a series of amides (3–11 and 13–18) related to
natural product 4-hydroxyisoleucine and screened their glucose
uptake activity in L6 rat muscle cells. Some of the compounds of
this series (6, 7, 8, 10 and 11) are more potent to the natural prod-
uct 4-HIL (1) in in vitro glucose uptake activity. Our preliminary
structure activity relationship data indicated that N-Boc protected
amides are more active than N-tosylated and N-acetylated amides
3-kinase-dependent pathway. Eur. J. Nutr. 51, 893–898.
Mitra, A., 2008. Some salient points in dietary and life-style survey of rural bengal
particularly tribal populace in relation to rural diabetes prevalence. Stud. Ethno-
Med. 2, 51–56.
Narender, T., Puri, A., Shweta Khaliq, T., Saxena, R., Bhatia, G., Chandra, R., 2006.
4-Hydroxyisoleucine an unusual amino acid as antidyslipidemic and antihyper-
glycemic agent. Bioorg. Med. Chem. Lett. 16, 293–296.
Nicolas, C., McNicol, P., Lucie Jette, L., 2006. Compounds and compositions for
use in the prevention and treatment of obesity and related syndromes, US
(
7 vs 13 and 8 vs 3).
20060223884.
Reaven, G.M., 1995. Pathophysiology of insulin resistance in human disease. Physiol.
Rev. 75, 473–486.
Conflict of interest
Sauvaire, Y., Petit, P., Broca, C., Manteghetti, M., Baissac, Y., Fernandez-Alvarez, J.,
Gross, R., Roye, M., Leconte, A., Gomis, R., Ribes, G., 1998. 4-Hydroxyisoleucine:
a novel amino acid potentiator of insulin secretion. Diabetes 47, 206–210.
The authors have no conflict of interest.
Disclosure statement
Shapiro, S.L., Parrino, V.A., Freedman, L., 1959. Hypoglycemic agents. III.1 N -Alkyl-
–3
1
and aralkylbiguanides. J. Am. Chem. Soc. 81, 3728–3736.
Shu, Y.Z., 1998. Recent natural products based drug development: a pharmaceutical
industry perspective. J. Nat. Prod. 61, 1053–1071.
Tamrakar, A.K., Jaiswal, N., Yadav, P.P., Maurya, R., Srivastava, A.K., 2011. Pongamol
from Pongamia pinnata stimulates glucose uptake by increasing surface GLUT4
level in skeletal muscle cells. Mol. Cell. Endocrinol. 339, 98–104.
Tamrakar, A.K., Schertzer, J.D., Chiu, T.T., Foley, K.P., Bilan, P.J.D., Philpott, J., Klip,
A., 2010. NOD2 activation induces muscle cell-autonomous innate immune
responses and insulin resistance. Endocrinology 15, 5624–5637.
The authors have nothing to disclose.
Acknowledgments
Authors are thankful to Director, CSIR-CDRI for constant encour-
agement on the program on natural products’ synthesis, SAIF for
spectral data, ICMR for ado project (No. 59/06/2010/BMS/TRM) and
fellowship to Mr. Rohit. This is CDRI communication No. 8817.
International Diabetes Federation, 2012. www.idf.org/diabetesatlas, International
Diabetes Federation. IDF Diabetes Atlas, 5th edition. International Diabetes Fed-
eration, Brussels (accessed 21.02.13).