Isoalantolactone derivative promotes glucose utilization in skeletal muscle cells and increases energy expenditure in db/db mice via activating AMPK-dependent signaling

Article abstract of DOI:10.1016/j.mce.2017.07.015

Augmenting glucose utilization and energy expenditure in skeletal muscle via AMP-activated protein kinase (AMPK) is an imperative mechanism for the management of type 2 diabetes. Chemical derivatives (2a-2h, 3, 4a-4d, 5) of the isoalantolactone (K007), a bioactive molecule from roots of Inula racemosa were synthesized to optimize the bioactivity profile to stimulate glucose utilization in skeletal muscle cells. Interestingly, 4a augmented glucose uptake, driven by enhanced translocation of glucose transporter 4 (GLUT4) to cell periphery in L6 rat skeletal muscle cells. The effect of 4a was independent to phosphatidylinositide-3-kinase (PI-3-K)/Akt pathway, but mediated through Liver kinase B1 (LKB1)/AMPK-dependent signaling, leading to activation of downstream targets acetyl coenzyme A carboxylase (ACC) and sterol regulatory element binding protein 1c (SREBP-1c). In db/db mice, 4a administration decreased blood glucose level and improved body mass index, lipid parameters and glucose tolerance associated with elevation of GLUT4 expression in skeletal muscle. Moreover, 4a increased energy expenditure via activating substrate utilization and upregulated the expression of thermogenic transcription factors and mitochondrial proteins in skeletal muscle, suggesting the regulation of energy balance. These findings suggest the potential implication of isoalantolactone derivatives for the management of diabetes.

Full text of DOI:10.1016/j.mce.2017.07.015

Accepted Manuscript
Isoalantolactone derivative promotes glucose utilization in skeletal muscle cells and
increases energy expenditure in db/db mice via activating AMPK-dependent signaling
Deepti Arha, E. Ramakrishna, Anand P. Gupta, Amit K. Rai, Aditya Sharma, Ishbal
Ahmad, Mohammed Riyazuddin, Jiaur R. Gayen, Rakesh Maurya, Akhilesh K.
Tamrakar
PII:
S0303-7207(17)30389-1
10.1016/j.mce.2017.07.015
MCE 10014
DOI:
Reference:
To appear in: Molecular and Cellular Endocrinology
Received Date: 21 February 2017
Revised Date: 16 June 2017
Accepted Date: 19 July 2017
Please cite this article as: Arha, D., Ramakrishna, E., Gupta, A.P., Rai, A.K., Sharma, A., Ahmad, I.,
Riyazuddin, M., Gayen, J.R., Maurya, R., Tamrakar, A.K., Isoalantolactone derivative promotes glucose
utilization in skeletal muscle cells and increases energy expenditure in db/db mice via activating AMPK-
dependent signaling, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.07.015.
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Isoalantolactone derivative promotes glucose utilization in skeletal muscle cells and
increases energy expenditure in db/db mice via activating AMPK-dependent signaling
a, d#
a
b#
c
a
a
Deepti Arha
, E. Ramakrishna , Anand P. Gupta , Amit K. Rai , Aditya Sharma ,
c
c
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Ishbal Ahmad , Mohammed Riyazuddin , Jiaur R. Gayen , Rakesh Maurya , Akhilesh K.
a, d
Tamrakar
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Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow-226031
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Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-
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226031
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Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-
226031
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Academy of Scientific and Innovative Research, New Delhi 110001,India.
#
Equal contribution
*Corresponding authors
Dr. Akhilesh Kumar Tamrakar
Division of Biochemistry, CSIR-Central Drug Research Institute
Sec-10, Jankipuram Extension, Sitapur Road, Lucknow-226031, India
E-mail: akhilesh_tamrakar@cdri.res.in
Tel: +91-0522-2772550 Ext. 4635
Fax: +91-0522-2771941
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Abstract
Augmenting glucose utilization and energy expenditure in skeletal muscle via AMP-activated
protein kinase (AMPK) is an imperative mechanism for the management of type 2 diabetes.
Chemical derivatives (2a-2h, 3, 4a-4d, 5) of the isoalantolactone (K007), a bioactive molecule
from roots of Inula racemosa were synthesized to optimize the bioactivity profile to stimulate
glucose utilization in skeletal muscle cells. Interestingly, 4a augmented glucose uptake, driven
by enhanced translocation of glucose transporter 4 (GLUT4) to cell periphery in L6 rat skeletal
muscle cells. The effect of 4a was independent to phosphatidylinositide-3-kinase (PI-3-K)/Akt
pathway, but mediated through Liver kinase B1 (LKB1)/AMPK-dependent signaling, leading to
activation of downstream targets acetyl coenzyme A carboxylase (ACC) and sterol regulatory
element binding protein 1c (SREBP-1c). In db/db mice, 4a administration decreased blood
glucose level and improved body mass index, lipid parameters and glucose tolerance associated
with elevation of GLUT4 expression in skeletal muscle. Moreover, 4a increased energy
expenditure via activating substrate utilization and upregulated the expression of thermogenic
transcription factors and mitochondrial proteins in skeletal muscle, suggesting the regulation of
energy balance. These findings suggest the potential implication of isoalantolactone derivatives
for the management of diabetes.
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Keywords: Insulin resistance, Isoalantolactone, Energy metabolism, Inula racemosa, Glucose
utilization.
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1. Introduction
Type 2 diabetes mellitus (T2DM) is characterized by abnormal insulin secretion and insulin
resistance, leading to derangements in carbohydrates, lipid and proteins metabolism (Kahn,
2003). Insulin resistance is a complex pathophysiological condition characterized by impaired
insulin action in metabolic tissues, including skeletal muscle, liver and adipose (Kahn et al.,
2006). From these tissues, skeletal muscle account for ~40% of the total body mass and is the
major site for glucose utilization and homeostasis (Engeli, 2012). A major pathogenic defect in
diabetes has been characterized by the impeded capacity of peripheral tissues, most notably the
skeletal muscle to utilize glucose effectively in face of hyperinsulinemia (Petersen and Shulman,
2006). This leads to simultaneous rise in blood glucose level and ends up with diabetes.
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Obesity is the major risk factor for the development of insulin resistance. Obesity
develops when energy intake exceeds energy expenditure. Increased energy retention due to lack
of expenditure predisposes an individual towards obesity which cause insulin resistance and
further T2DM (Tseng et al., 2010). While current obesity management is focused on reducing
caloric intake, increasing energy expenditure has been suggested as an alternative approach for
the disease management. This imply specially in case of adaptive thermogenesis in which energy
is released in the form of heat and occurs primarily in the mitochondria of skeletal muscle and
brown adipocytes (Tseng et al., 2010). Skeletal muscle is an important tissue for exercise-
induced thermogenesis (Himms-Hagen, 2004). In skeletal muscle, AMP-activated protein kinase
(AMPK) acts as a major modulator of fuel preference and energy metabolism (Hardie et al.,
2012). Pharmacological activation of AMPK imparts pseudo effect of exercise and is beneficial
for patients with T2DM (Winder and Hardie, 1999). Activation of AMPK results in stimulation
of glucose uptake inside the muscle cell driven by enhanced rate of translocation of glucose
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transporter 4 (GLUT4) to the cell surface and inhibit glucose output from liver (Hardie et al.,
2013; Huang and Czech, 2007). At the same time, chronic activation of AMPK enhances
mitochondrial function in skeletal muscle (Koh et al., 2008). Therefore, interventions with ability
to activate AMPK in skeletal muscle can modulate glucose utilization and energy metabolism as
well for the management of T2DM.
Traditional medicines from plants have an enormous potential to cure various diseases
and have been most valuable source of drug leads and to provide useful structural architectures
that feature selective and potential access to new biological targets (Newman and Cragg, 2012).
Eudesmanolide type sesquiterpene lactones are an excellent example of natural products with
wide variety of activities (Di et al., 2014; Chen et al., 1994). They built from three isoprene units
and characterized by the presence of exomethylene/methyl at C-4 and C-11 position. They have
been reported to show wide range of biological activities, like antiproliferative, anti-
inflammatory (Lajter et al., 2014), antiviral, antimicrobial (Ciric et al., 2012), cytotoxic (Rosselli
et al., 2012), and lipid lowering activities (Jiang et al., 2011). Isoalantolactone is one among
eudesmanolide type sesquiterpene lactones. This compound is widely distributed in Inula
species, and revealed to possess cytotoxic, anti allergic, anti cancer, anti mycobacterial and
antioxidant activities (Xiang et al., 2016; Cantrell et al., 1999; Seo et al., 2009, Li et al., 2016).
As part of our ongoing research program in the area of natural products for the
management of diabetes, we explored roots of Inula racemosa, an ornamental plant, widely
distributed in India, China and Europe. This plant is a rich source of eudesmanolide type
sesquiterpene lactones, in particular isoalantolactone and alantolactone (Ma et al., 2013). Ajani
et. al. (2009) has reported the anti diabetic activity of the methanolic extract of I. racemosa, but
the precise active ingredient responsible for the antidiabetic activity and the modes of action of
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this plant are largely unknown. Here, we investigated the chemical constituents of I. racemosa
and isolated isoalantolactone (1, K007) as a potent stimulator of glucose transport in skeletal
muscle cells. Further, we synthesized fourteen derivatives (2a-2h, 3, 4a-4d, 5) of K007 to
optimize its bioactivity. Derivatization of isoalantolactone was done by N-substistuted
piperizines on C-13 position, allylic hydroxylation followed by esterification at C-3 position. All
the derivatives were initially evaluated for effect on glucose uptake in skeletal muscle cells and
the active one was subjected to detail mechanistic analysis and in vivo efficacy to regulate
glucose homeostasis in db/db mice. We report that isoalantolactone derivative 4a stimulates
glucose utilization in clonal skeletal muscle cells and exerts potent antidiabetic effects through
modulating energy expenditure in db/db via LKB1/AMPK-dependent signaling.
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2. Materials and Methods
2.1. Materials
Cell culture medium (DMEM), antibiotic/antimycotic solution, fetal bovine serum, and trypsin
were from Gibco, USA. 2-deoxyglucose, O-phenylenediamine dihydrochloride, protease
inhibitor cocktail, polyclonal anti-myc, monoclonal anti-actinin-1, dosromorphin, wortmannin
and all other chemicals unless otherwise noted were from Sigma Chemical (St. Louis, MO). 2-
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Deoxy-D-[ H]-glucose (2-DG) was from MP Biomedicals. Antibodies to phospho-Akt (Ser-
473), Akt, phospho-AMPKα (Thr-172), AMPKα, phospho-ACC (Ser-79), phospho-LKB1 (Ser-
428), LKB-1, p-SREBP-1c (Ser-372), COX IV, Cytochrome C, hexokinase II and GLUT4 (1F8)
were from Cell Signaling Technology (USA). Antibody to GLUT1 was from Santa Cruz
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Biotechnology, Inc (USA). Triglycerides and total cholesterol measurement kits were from
Dialabs and insulin level was measured by mouse insulin ELISA kit (RayBiotech Inc).
2.2. Extraction and isolation of isoalantolactone (K007)
The roots of Inula racemosa were procured from local market and identified in the Botany
Division of CDRI. The voucher specimen (3853) is preserved in the herbarium of the Institute
for future reference. The air dried powdered roots were extracted five times with ethanol at room
temperature. The combined EtOH extract was filtered and concentrated under vacuum, which
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afforded a dark brown residue (C002). The crude ethanolic extract was dissolved in distilled H O
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and fractionated with n-hexane (F003), chloroform (F004) and n-butanol (F005) to afford
respective solvent fractions. Based on bioactivity, the n-hexane fraction was taken up for a detail
chemical investigation. The fraction (F003) was dissolved in chloroform, adsorbed over silica gel
(60-120 mesh, 220 g) and subjected to column chromatography (60-120 mesh, 4.0 Kg). The
column was eluted with a mixture of hexane: ethyl acetate (96:04) and afforded a white
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crystalline compound (1, K007), which was characterized as isoalantolactone by H, C, IR
spectral data.
2.3. Cell culture
L6 skeletal muscle cells and L6 cells stably expressing rat GLUT4 with a myc epitope inserted in
the first exofacial loop (L6-GLUT4myc), a kind gift of Dr. Klip, Program in Cell Biology, The
Hospital for Sick Children, Toronto, Canada were maintained in DMEM supplemented with 10%
FBS and 1% antibiotic/antimycotic solution in a humidified atmosphere of air and 5% CO
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at
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37 C. Differentiation was induced by switching confluent cells to medium supplemented with
2% FBS. Experiments were performed in differentiated myotubes 5-6 days after seeding.
2.4. Glucose uptake measurement
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Glucose uptake inside the myotubes was measured using [ H] 2-deoxy-D-glucose (2-DG), a non
metabolizable analog of D-glucose, as the substrate as described previously (Tamrakar et al.,
2010). Briefly, myotubes were treated as indicated with final 3h in serum-deprived medium and
a sub-set of cells were stimulated with 100 nM insulin for 20 min. Myotubes were then incubated
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in HEPES-buffered saline supplemented with 10 µM 2-DG (0.5 µCi/ml 2-[ H] DG) for 10 min at
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room temperature, followed by cell lysis and measurement of radioactivity incorporated by
scintillation counting. Nonspecific uptake was determined in the presence of cytochalasin B (50
µM) during the assay. Glucose uptake measured in triplicate and normalized to total protein, was
expressed as fold induction with respect to unstimulated cells.
2.5. GLUT4 translocation measurement
GLUT4 translocation to cell surface was determined in L6-GLUT4myc myotubes by measuring
the cell surface level of GLUT4myc by an antibody-coupled colorimetric assay as previously
described (Tamrakar et al., 2011). The fraction of GLUT4myc at the cell surface, measured in
triplicate, was expressed as fold induction with respect to unstimulated cells.
2.6. Assessment of cell viability
Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) assay (Mosmann, 1983). After indicated treatments, the cells were incubated with MTT
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solution (5 mg mL in PBS) for 4h and the absorbance was measured at 540 nm using an ELISA
plate reader (Bioteck, USA).
2.7. Western analysis
After indicated treatments, myotubes or tissue samples were lysed in RIPA buffer supplemented
with protease and phosphatase inhibitors. Lysates were cleared by centrifugation at 10000 rpm
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for 10 min at 4 C and protein content was measured by the BCA assay. For western blotting,
proteins were boiled in Laemmli buffer, separated by SDS-PAGE and transferred onto PVDF
membrane. Membranes were then blotted using primary antibodies (4°C overnight), washed and
peroxidase-coupled secondary antibody was applied for 1h at room temperature. Membranes
were developed using enhanced chemiluminescence (ECL, Millipore), and analyzed using NIH
Image J software.
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2.8. Determination of cellular ATP levels
The cellular ATP levels were estimated in L6 myotubes by Stay Brite ATP assay Kit (Biovision)
as per manufacturer’s protocol. Briefly, after the indicated incubation, harvested cells were lysed
in the reaction buffer and cellular debris was removed by centrifugation. The cellular level of
ATP was determined by mixing the supernatant with luciferase reagent that catalysed the light
production from ATP and luciferin. Luminescence was measured by a luminometer. Protein
concentrations were measured using BCA protein assay kit. The ATP level in each sample was
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expressed as pmol µg protein.
2.9. Animals
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Male C57BL/KsJ-db/db mice (40±5 g) used for the study were kept in polypropylene cage in
group of 5-6 animals/cage. The work with these animals was cleared by Institutional Animal
Ethics Committee (IAEC) and was conducted in accordance with the guidelines of the
Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA)
formed by the Government of India. Mice were housed under standard conditions of temperature
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23 ±2 C with relative humidity (50–60%), light 300 Lx at floor level along with light and dark
cycles of 12h. Animals were provided with standard diet and drinking water ad libitum.
2.10. Antidiabetic efficacy in db/db mice
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The animals were divided into groups of six animals each. Group I was regarded as the control
group and treated with vehicle, whereas the remainder was termed as compound-treated groups
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and dosed orally at the doses of 3,10 and 30 mg kg of compound or metformin (250 mg kg )
for 15 days. Blood glucose level and body weight of each animal was measured at two days
interval. On day 15, an oral glucose tolerance test (OGTT) of each animal was performed after
an overnight fast. The baseline blood glucose level was monitored at 0 min, followed by an oral
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glucose load of 3 g kg body weight. The blood glucose levels were again checked at 30 min, 60
min, 90 min, and 120 min post-glucose administration, using a glucometer. Blood had withdrawn
from the retro-orbital plexus of the eye for the estimation of serum lipid profile and insulin.
2.11. Comprehensive laboratory animal monitoring system (CLAMS)
The Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments,
Columbus, OH) is a set of live-in cages for automated, non-invasive and simultaneous
monitoring of horizontal and vertical activity, feeding and drinking, oxygen consumption and
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CO2 production. The db/db mice treated with compound, along with control animals were
individually kept in CLAMS cages with a 12-h light/12-h dark cycle in an ambient temperature
of 22-24°C, and monitored over 3-day period. Mice were kept conscious and unrestrained during
the measuring period. Food and water consumption are measured directly as accumulated data.
VO and VCO rates were determined under Oxymax system settings as follows: air flow, 0.8 L
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min ; sample flow, 0.4 L min . The expired air was analyzed for a 5 sec period every 7 min
using an electrochemical O analyzer and a CO sensor. The system was calibrated against a
standard gas mixture to measure O consumed (VO
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were evaluated over a 3-day period. Energy expenditure (kcal kg h ; heat production) was
calculated using a rearrangement of the Weir equation as supplied by Columbus Instruments:
(3.815+1.232*RER)*VO . A mean of 100 determined values per mice was averaged for the final
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value of each dark/light cycle. Resting VO2 measured during none to less than baseline activity
(0–10 counts/10 min) served as a measure of basal or resting metabolic rate.
2.12. Tissue triglyceride and total cholesterol measurement
After the treatment period, mice were sacrificed, and the liver and skeletal muscle tissue sample
were excised and frozen in liquid nitrogen for further analysis. Triglyceride levels were
measured in 150 mg tissue sample homogenized in 300µl of homogenization buffer (18 mM
Tris, pH 7.5, 300 mM mannitol, and 50 mM EGTA). After homogenization, 5ml of chloroform-
methanol mixture (2:1) was added and the sample was incubated at room temperature for an hour
with occasional vortexing. Post incubation, 1ml of Milli-Q water was added to the sample, mixed
thoroughly and centrifuged at 4,700 rpm at 4ºC. The lower organic phase was carefully collected
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in a glass tube and solvent was evaporated in nitrogen dryer (Hall et al., 2014). Dried samples
were resuspended in 0.5% Triton X-100 by sonication. To estimate triglyceride in the sample,
10µl of this mixture was added to the 1ml of triglyceride reagent (Dialab) and incubated for 10
minute at 37ºC followed by measurement of optical density at 500 nm. Total cholesterol level
was measured with Dialab reagent in similar way as triglycerides.
2.13. RNA extraction and gene expression analysis
Total RNA was extracted from the tissue by isothiocyabate/phenol/chlorofrom (Trizol) method.
Complementary DNA was prepared using Verso cDNA synthesis kit (Thermo Scientific). For
semi-quantitative analysis cDNA was amplified by PCR using gene specific primers. 18S rRNA
was taken as internal loading control. List of primers is given in table 1.
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2.14. Statistical analysis
Values are given as mean ± SEM. Analysis of statistical significance of differences in
measurements between samples was done by two-way ANOVA or one-way ANOVA with
Dunnets post hoc test (GraphPad Prism version 3). P<0.05 was considered statistically
significant.
3. Results
3.1. Effect of K007 on glucose uptake in skeletal muscle cells
Glucose uptake inside the skeletal muscle cells is the rate limiting step in its utilization. In an
attempt to identify natural molecules that could stimulate glucose uptake like insulin, activity
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guided fractionation of the ethanolic extract of the roots of I. racemosa led to the identification
of an isoalantolactone, K007 (1, Fig. 1A) as a potential stimulator of glucose uptake in skeletal
muscle cells. Incubation of L6 myotubes with K007 significantly increased basal glucose uptake,
with 1.4- and 2.5-fold (p<0.01) stimulation at 2.5 and 5.0 µM concentrations, respectively (Fig.
1B). The effect of K007 on insulin-stimulated glucose uptake was determined by incubating the
cells with K007, followed by acute insulin treatment (100 nM, 20min). Insulin alone caused
1.46-fold (p<0.05) increase in glucose uptake; prior treatment with K007 resulted in an increase
of insulin response in an additive manner (Fig. 1B). The effect of K007 on glucose uptake in L6
myotubes was found to be concentration-dependent at 2.5 and 5.0 µM, however further increase
in concentration lead to drastic decrease in the rate of glucose uptake (Fig. 1B). Therefore, we
evaluated the effect of K007 on viability of L6 myotubes by MTT. Overnight treatment with
lower doses of K007 (2.5 and 5.0 µM) had no significant effect on cell viability; further increase
in concentration significantly decreased the viability of L6 myotubes in a concentration-
dependent manner (Fig. 1C).
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3.2. Synthesis and the effect of K007 derivatives on glucose uptake in muscle cells
To ward off the deleterious effect of the K007 and optimize its efficacy as a stimulator of
peripheral glucose utilization, a series of derivatives (2a-2h, 3, 4a-4d, 5) were synthesized with
the structural modification on the basic scaffold. Michael addition is the most frequent reaction
to derivatize α, β-unsaturated carbonyl compounds, hence we chose the reaction, to understand
the role of exocyclic double bond present at C-11 position, with N-substituted piperazines to give
Michael addition products (2a-2h, Fig. 1, Scheme 1) and other exocyclic double bond at C-4 was
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reacted with Sc(OTf) to form the alloalantolactone (5, Fig. 2, Scheme 2) which is an analogue
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of isoalantolactone formed by 1,3-H shift. Our further approach for the chemical transformation
of K007 was allylic hydroxylation followed by esterification using SeO , DCC, DMAP and
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different type carboxylic acids to yield respective esters (4a-4d, Fig. 2, Scheme 3). Structural
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elucidation of the derivatives was performed using H, C, IR spectral data.
In order to determine the potential activity of these derivatives, we analyzed their effects
to stimulate glucose uptake in muscle cells. L6 myotubes were incubated with derivatives at 5
µM concentration for overnight period and glucose uptake was measured. Results depicted in the
Table 2 illustrate that different derivatives showed diverse efficacy to modulate the rate of
glucose uptake in L6 myotubes. The most significant increase in glucose uptake was observed
with derivatives 4a, 4b and 4c (3.5-, 2.2- and 2.5-fold, respectively, P<0.01). Among them, 4a
was found to be most active, and evaluated for the effect on cell viability. In contrast to the
parent compound (K007, 1), 4a did not exert any significant effect on cell viability at the
concentrations ranged from 2.5 µM to 50 µM, after an overnight incubation, as assessed by the
MTT assay (Fig. 3C) and considered safe at these treatment conditions.
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3.3. Concentration- and time-dependent effect of 4a on glucose uptake
Treatment with 4a increased glucose uptake in L6 myotubes in a concentration-dependent
manner (Fig. 3A) with maximum stimulation of basal glucose uptake at 5.0 µM (3.2-fold,
p<0.001). Further increase in concentration did not cause any further augmentation in the rate of
glucose uptake, indicating the saturation level. Furthermore, 4a was found to exert a dose-
dependent increase of insulin response in an additive manner. Significant response was observed
from 5 µM concentration of 4a (Fig. 3A: control, 1.9-fold vs. 4a, 4.6-fold, p<0.001).
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Incubation of L6 myotubes with 4a (5 µM) resulted an increase in the rate of glucose
uptake in a time-dependent fashion under both basal and insulin-stimulated conditions (Fig. 3B)
with significant stimulation of basal glucose uptake after 6h. Maximum stimulation by 4a was
observed after 16 h, and in all subsequent experiments 16 h incubation was used.
3.4. Effect of 4a on GLUT4 translocation to the cell surface in muscle cells
Enhanced translocation of GLUT4 to cell periphery is an essential mechanism for inducible
glucose uptake inside the cells. To investigate the glucose uptake stimulatory effect, 4a was
probed for its ability to enhance GLUT4 translocation in L6-GLUT4myc myotubes. Consistent
with glucose uptake data, overnight incubation with 4a significantly enhanced the surface
GLUT4myc level in a concentration-dependent fashion (1.3- and 1.9-fold increase at 2.5 and 5
µM, respectively). Insulin alone resulted in a profound increase in surface GLUT4myc level
(1.75-fold vs. basal, p<0.001). Treatment of myotubes with 4a for 16 h with insulin added for
final 20 min, resulted in dose-dependent increase of insulin response in an additive manner
(p<0.001, Fig. 3D ).
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We next investigated whether the 4a-mediated raise in surface level of GLUT4myc was
accompanied by an increase in total amount of GLUT4. Cellular amount of GLUT4 was assessed
after overnight incubation. 4a did not affect the cellular amount of GLUT4 either under basal or
insulin-stimulated condition in L6 myotubes. Additionally, treatment with 4a had no effect on
the cellular amount of GLUT1, another major glucose transporter responsible for basal glucose
uptake in skeletal muscle cells (Fig. 3E and 3F).
3.5. Effect of 4a on PI-3-Kinase-dependent signaling pathway
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To investigate the involvement of pathways that contributed to GLUT4 translocation in skeletal
muscle, we mapped the effect of 4a on phosphatidylinositide-3-kinase (PI-3-K) activation, the
basic pathway responsible for the metabolic effects of insulin. The effect of wortmannin, a
selective inhibitor of PI-3-Kinase pathway (Malide et al., 1997) on 4a-stimulated glucose uptake
was examined in L6 myotubes. As represented in the Fig. 4A, insulin-stimulated glucose uptake
was completely abolished by wortmannin (p<0.01), but there was no significant effect on 4a-
stimulated glucose uptake in presence of wortmannin. These results suggest that the signal
transduction leading to glucose uptake by 4a is primarily mediated via a PI-3-K-independent
pathway. To further validate the finding, we evaluated the phosphorylation of Akt, a downstream
key note of PI-3-K pathway, at the regulatory sites Ser-473 and Thr-308. As expected, insulin
(100 nM) induced strong phosphorylation of Akt, but 4a did not cause any significant effect on
Akt phosphorylation, and consistent to glucose uptake data, presence of wortmannin profoundly
inhibited insulin-stimulated phosphorylation of Akt at Ser-473 and Thr-308 in L6 myotubes (Fig.
4B-D), verifying the action of wortmannin to inhibit PI-3-K mediated signaling.
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3.6. Effect of 4a on AMPK-dependent pathway
Given the glucose uptake stimulation by 4a was independent of PI-3-K/Akt pathway; we studied
its effect on the AMPK, a sensor of cellular energy status and established to stimulate glucose
uptake independent to insulin (Gowans et al., 2013). L6 myotubes were incubated with 4a (5µM)
or established AMPK activator metformin (500µM) in presence of dorsomorphin, a selective
inhibitor of AMPK. Dorsomorphin (20 µM) was added 30 min prior to treatment with 4a or
metformin. As shown in Fig. 5A, presence of dorsomorphin inhibited both 4a- and metformin-
stimulated glucose uptake to a significant level (p<0.01). These data suggest that 4a, like
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metformin, utilizes an AMPK-dependent pathway to mediate glucose uptake. To confirm the
activation of AMPK, we examined the effect of 4a on phosphorylation of AMPKα at Thr-172,
the principle regulatory site of AMPK activation (Hawley et al., 1996). 4a substantially increased
the phosphorylation of AMPKα (Thr-172), in a concentration-dependent manner, without having
any significant effect on total cellular content of AMPKα (Fig. 5B)
The major upstream kinase responsible for phosphorylation of AMPKα at Thr-172 is
liver kinase B1 (LKB1). To map whether 4a-mediated activation of AMPKα was LKB1-
dependent, we analyzed the effect of 4a on phosphorylation of LKB1 (Ser-428). Treatment with
4a augmented the phosphorylation of LKB1 (Ser-428) in a concentration-dependent manner
(Fig. 5C). These findings suggest that 4a utilizes LKB1/AMPK-dependent signaling to stimulate
glucose uptake in skeletal muscle cells. AMPK has been established as cellular fuel gauge,
activating its kinase activity with decrease in cellular energy charge (Winder and Hardie, 1999).
Treatment with 4a significantly decreased the cellular ATP level in L6 myotubes (Fig. 5D),
pointing the activation of cellular processes require energy expenditure.
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3.7. Effect of 4a on signalling downstream to AMPK
Besides enhancing glucose uptake inside the cells, AMPK also modulate fatty acid metabolism
by directly phosphorylating and inactivating its major downstream target acetyl CoA carboxylase
(ACC) at Ser-79 (Munday et al., 1998; Davies, 1990). Hence, to study the effect of 4a on
downstream signalling to AMPK, we assessed the phosphorylation of ACC (Ser-79). As
expected, 4a enhanced the phosphorylation of ACC, compared to control (Fig. 5E, p<0.05),
validating the activation of AMPK by 4a. Another downstream target of interest was sterol
response element binding protein-1c (SREBP-1c), an important lipogenic transcription factor
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regulated by exercise, nutritional modification and insulin (Horton et al., 2002). AMPK has been
known to phosphorylate SREBP-1c at Ser-372, thereby preventing its proteolytic cleavage and
translocation to nucleus and thus attenuating transcription of lipogenic genes (Li et al., 2011). In
L6 myotubes, 4a treatment elevated SREBP-1c (Ser-372) phosphorylation to a significant level
(Fig. 5F, P<0.05), suggesting that treatments with 4a may decrease the cellular lipid content.
3.8. Metabolic effects of 4a in db/db mice
The metabolic effects of 4a were further evaluated in diabetic db/db mice by administering the
compound for 15 days at different doses. There was no significant effect on body weight, but the
mean blood glucose of the treated animals decreased gradually, during the course of treatment
(Fig. 6 A & B). After the treatment period, animals were subjected to whole body composition
analysis by Echo-MRI. Treatment with 4a significantly reduced the body fat mass at the dose of
30 mg/kg (p<0.05), associated with an increase in the lean mass (p<0.05). Metformin treatment
had no significant effect on body mass composition of db/db mice (Fig. 6G). The mice treated
with 4a showed better tolerance to glucose load in a dose-dependent fashion (Fig. 6C). At the
dose of 30 mg/kg, 4a administration caused 35.2% (p<0.05) improvement in glucose tolerance,
which is quite comparable to physiological dose of metformin (250 mg/kg), causing 42.4%
improvement (p<0.01) (Fig. 6D). Skeletal muscle is the major depot for post parandial glucose
disposal thereby regulating glucose homeostasis. Treatment with 4a enhanced the gene
expression of GLUT4 and hexokinase, associated with increased GLUT4 protein level, but
unaltered hexokinase protein level in skeletal muscle of db/db mice (Fig. 6 E, F, H & I).
However, 4a did not have any significant effect on serum insulin level, whereas metformin
caused significant reduction in serum insulin level of db/db mice (Fig. 6J).
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To verify the participation of AMPK in 4a-mediated improvement in glucose
metabolism, we checked the activation of AMPK in skeletal muscle of db/db mice. AMPK
signaling was up regulated as confirmed by the increased phosphorylation of AMPK (Thr-172),
ACC (Ser-79) and LKB1 (Ser-428) in skeletal muscle of 4a treated mice, compared to control
(Fig. 7 A-D). Metformin also significantly up regulated the phosphorylation the AMPK and
associated enzymes. Metformin has been reported to increase the phosphorylation of AMPK in
liver (Shaw et al., 2005). To verify the effect of 4a on liver AMPK signaling, we assessed the
phosphorylation in liver of db/db mice. Similar to metformin, treatment with 4a significantly
enhanced the phosphorylation of AMPK (Thr-172) and ACC (Ser-79) in liver of db/db mice
(Fig. 7 E-G), suggesting the activation of AMPK signaling in liver.
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In addition to the beneficial effect on glucose metabolism, activation of AMPK has been
shown to exert favorable effect on lipid metabolism through phosphorylation and inhibition of
enzymes involved in cholesterol and lipid synthesis (Muoio et al., 1999). Therefore, we
investigated the effect of 4a treatment on serum and tissue levels of cholesterol and triglycerides
in db/db mice. Similar to the metformin, treatment with 4a resulted in a significant decrease in
serum and hepatic level of total cholesterol in a dose-dependent fashion (Fig. 7 H & I). Serum
triglyceride level was also decreased by lower dose of 4a (10 mg/kg) or metformin, but there was
significant elevation in serum triglyceride level upon treatment with 30 mg/kg dose of 4a in
db/db mice (Fig. 7J). In contrast, the hepatic and skeletal muscle triglyceride levels were
significantly decreased by 4a in db/db mice (Fig. 7 K & L). We further assessed the effect of 4a
on liver lipid metabolism by measuring the expression of key target genes of lipid metabolism in
liver of db/db mice. Treatment with 4a did not cause any significant change in gene expression
of ACC, but there was a significant decrease in Stearoyl-CoA desaturase (SCD1). Metformin
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treatment did not cause any significant effect on SCD1 expression. Moreover, both 4a and
metformin reduced the expression of SREBP-1c and PPAR-α (Figure 8).
3.9. Effect of 4a on energy homeostasis in db/db mice
Energy homeostasis is maintained by regulating a delicate balance between energy intake and
energy expenditure. Increase in energy intake will lead to obesity as in the case of type 2
diabetes, so is the lack of expenditure. We analyzed the effect of 4a (30 mg/kg) on energy
expenditure using comprehensive lab animal monitoring system (CLAMS). The db/db mice
treated with 4a showed increased oxygen consumption and carbon dioxide production rate than
the control animals through a 12h light/dark cycle (Fig. 9 A-D), suggesting the improvement in
metabolic profile of the treated animals. Although, there was no significant change in respiratory
exchange ratio (RER), implement that the mice were still using the carbohydrate as source of
energy (Fig. 9 E & F). The db/db mice treated with 4a showed significant increase in whole body
energy expenditure (in form of heat) with respect to control animals (Fig. 9 G & H). This
increase in energy expenditure was not due to the increase in physical activity as revealed by the
unaffected x axis and z axis movement (Fig. 10 A & B). There was a significant improvement in
basal metabolic rate and resting metabolic rate of 4a-treated db/db mice, clearly indicating an
increase in energy expenditure (Fig. 10 C & D). On the other hand, treatment of 4a in db/db mice
showed an increase in food intake in comparison to control animals (Fig. 10E). But there was no
significant difference in body weight of treated animals compared to control mice.
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The 4a treatment induced the activation of a network of genes regulating energy
homeostasis and mitochondrial functions in skeletal muscle of db/db mice. The expression of the
transcription factors, including PPARα, PGC-1α, NRF1, and Dio2, was increased in skeletal
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muscle of the 4a treated animals (Fig. 11 A & B). The expression of UCP1 and other
mitochondrial marker genes such as ATP synthase, COX IV, and Cytochrome C, was also
significantly induced (Fig. 11 C & D). Western blot analysis indicated the enhanced protein level
of COX IV and Cytochrome C by 4a treatment in skeletal muscle tissue of db/db mice (Fig. 11
E-G). These lines of evidence suggest that 4a could enhance energy expenditure by regulating
mitochondrial functions in skeletal muscle of db/db mice.
4. Discussion
Skeletal muscle is the prominent situate for maintenance of postprandial glucose homeostasis
and is the primary tissue contributing to whole-body energy expenditure. In skeletal muscle
glucose metabolism is mainly regulated by insulin, which exerts its metabolic actions by
activating signaling cascade involving insulin receptor substrate (IRS), PI3K, and Akt, leading to
the translocation of GLUT4 from intracellular pool to cell membrane, where they facilitate
glucose transport inside the cells (Pessin et al., 1999). Skeletal muscle insulin resistance, the
impaired response of insulin to regulate glucose metabolism, is a major factor in the
pathogenesis of T2DM (Yu et al., 2002). However, various other stimuli such as osmotic shock
(Chen et al., 1997), activation of G-protein coupled receptors (Wang et al., 2000), contraction in
skeletal muscle (Yeh et al., 1995) have been reported to induce insulin-like effect to stimulate
glucose utilization through insulin-independent pathways. Therefore, targeting insulin
independent mechanisms to regulate glucose metabolism may be helpful in management of
T2DM.
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Obesity represents a major risk factor for the development of insulin resistance and other
features of the metabolic syndrome. Obesity develops when there is an imbalance between the
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energy intake and energy expenditure, and targeting energy expenditure has proven to be an
attractive concept for managing obesity and associated metabolic complications (Tseng et al.,
2010). Adaptive thermogenesis, a physiological process whereby energy is dissipated in the form
of heat in response to external stimuli provides a mean to target energy expenditure. Skeletal
muscle is an important tissue for thermogenesis (Himms-Hagen, 2004). In skeletal muscle
AMPK is the central enzyme to regulate cellular bioenergetics, which senses the nutrient status
and help to regulate glucose transport, fatty acid oxidation, and metabolic adaptations in skeletal
muscle (Koh et al., 2008). Therefore, AMPK activation in skeletal muscle can regulate both the
glucose metabolism and cellular bioenergetics, benefecial for the management of metbolic
complications, including T2DM.
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In an attempt to discover small molecule from natural sources that could stimulate
glucose uptake, isoalantolactone (K007) from the roots of I. racemosa was identified with
potential to augment glucose uptake in L6 myotubes. However, noticing the adverse effect of
K007 on cell viability at higher concentration, chemical derivatives of K007 were synthesized to
optimize its bio-efficacy without cytotoxic effect. Among the derivatives, 4a displayed potent
stimulation of glucose uptake in L6 myotubes in a concentration- and time-dependent fashion,
without affecting the cell viability. The effect of 4a (5.0 µM) was significantly higher than that
of insulin (100 nM) at concentration that produced maximum response. Similar to insulin, 4a
treatment enhanced the rate of GLUT4 translocation to cell surface in skeletal muscle cells,
required for the entry of glucose inside the cells (Huang and Czech, 2007). It also potentiated
insulin-stimulated glucose uptake and GLUT4 translocation in an additive manner, suggesting
that 4a-induced signaling leading to glucose uptake is distinct from that induced by insulin.
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The metabolic actions of insulin are mediated through activation of PI-3-K (Taniguchi et
al., 2006). Presence of wortmannin, a specific inhibitor of PI-3-K abolished the insulin mediated
stimulation of glucose uptake to basal level. However, in case of 4a, wortmannin had no effect
on glucose uptake stimulation, negating the participation of PI-3-K. Downstream to PI-3-K, Akt
has been shown to be involved in insulin stimulated glucose uptake in skeletal muscle cells
(Taniguchi et al., 2006). Insulin induced a strong phosphorylation of Akt at Ser-473 and Thr-308,
but 4a did not induce any marked enhancement of Akt phosphorylation. These findings confirm
that 4a stimulated glucose uptake is primarily mediated via a PI-3-K/Akt-independent signaling
pathway. The possible candidate for such PI-3-K independent stimulation of glucose uptake
might be the AMPK, which is an intracellular fuel sensor and has been shown to play a role in
the physiological control of insulin-independent glucose uptake (Rutter et al., 2003). AMPK
activation has been established to increase glucose uptake during exercise via stimulating
GLUT4 translocation in skeletal muscle cells. Several plant derived bioactive molecules,
including metformin have been reported to enhance glucose utilization through AMPK-
dependent signaling pathway (Lee et al., 2006; Shaw et al., 2005, Shen et al., 2014). Presence of
dorsomorphin, a specific inhibitor of AMPK, inhibited both 4a and metformin-induced glucose
uptake in L6 myotubes, suggesting the participation of AMPK-dependent signaling pathway.
AMPK is activated by phosphorylation of Thr-172 residue of its catalytic α subunit (Hawley et
al., 2003) by upstream kinase LKB1. The LKB1 kinase is a tumor suppressor protein
ubiquitously expressed in mammalian cells and has been shown to phosphorylate and activate
AMPK in response to metformin and energy depleted condition (Shaw et al., 2004, Shaw et al.,
2005). We also confirmed an increase in the phosphorylation of AMPK (Thr-172) by 4a in a
concentration-dependent manner, indicating the activation of AMPK. It has been reported that
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phosphorylation of LKB1 at Ser-428 lead to nuclear export of LKB1 from cytosol, which in turn
lead to the phosphorylation of AMPKα (Xie et al., 2008). The 4a augmented the phosphorylation
of LKB1 (Ser-428), pointing the activation of LKB1/AMPK axis. Besides phosphorylation,
AMPK is also activated by metabolic stress associated with decreased cellular ATP level. Under
such condition AMP and ADP levels increases, leading to allosteric activation of AMPK
(Oakhill et al., 2011). Here, treatment with 4a significantly reduced the cellular ATP level in L6
myotubes, indicating the change in cellular bioenergetics, favoring the activation of AMPK. Data
indicate that both activation of LKB1 and decrease in cellular ATP level might participate
independently of interdependently to activate AMPK in response to 4a. These results strongly
suggest that activation of AMPK signaling by 4a in skeletal muscle cells up regulate glucose
uptake, independent to insulin signaling.
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Activation of AMPK has correlated with its kinase activity resulting in phosphorylation
and inhibition of ACC, the rate limiting enzyme controlling fatty acid synthesis. Phosphorylation
of ACC accounts for both allosteric and covalent modification components of AMPK activation
(Gowans et al., 2013). Consistent to the effect on AMPK, 4a significantly enhanced the
phosphorylation of ACC (Ser-79). Another important target of AMPK related to lipid
metabolism is the SREBP. SREBP-1c is a nutritionally regulated lipogenic transcription factor
involved in activation of genes of fatty acid and triglyceride synthesis (Horton et al., 2002).
AMPK directly interact and stimulate the phosphorylation of SREBP-1c (Ser-372), resulting in
the suppression of its cleavage and nuclear translocation to regulate gene expression (Li et al.,
2011). Treatment with 4a resulted in increased phosphorylation of SREBP-1c (Ser-372) in L6
myotubes. It is conceivable from this data that this pathway would play an important role in
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regulating lipogenesis in other tissues such as adipose and liver, contributing to improved
systemic response.
Inspired from the in vitro effects of 4a on glucose metabolism in L6 myotubes, we
examined the effect of 4a on glucose homeostasis in db/db mice, a well-established model for
type 2 diabetes. The administration of 4a for 15 days led to increased phosphorylation of AMPK
(Thr-172), ACC (Ser-79) and LKB1 (Ser-428) in skeletal muscle of db/db mice, validating the in
vitro effect of 4a on AMPK signaling. Additionally, there was an increase in the phosphorylation
of AMPK (Thr-172) and ACC (Ser-79) in liver of db/db mice treated with 4a or metformin,
suggesting the activation of AMPK signaling in liver. Furthermore, treatment with 4a resulted in
dose-dependent decrease in blood glucose level, without having any significant effect on body
weight. The 4a treated animals displayed a better tolerance to glucose load, which was
particularly prominent and comparable to metformin, at the dose of 30 mg/kg. It is worthy of
noticing that 4a stimulates GLUT4 translocation in skeletal muscle cells leading to enhanced
glucose uptake, which might be responsible for the improved glucose tolerance in animal system.
Moreover, chronic treatment with 4a was associated with increased expression of the GLUT4 in
skeletal muscle of the db/db mice, contributing to improved glucose tolerance. In support to our
data, chronic activation of AMPK by AICAR has also been shown to increase skeletal muscle
GLUT4 expression in normal rats (Holmes et al., 1999), postulating the ability of chronic AMPK
activation to upregulate the proteins that mediate glucose uptake in skeletal muscle, to modulate
whole body glucose metabolism.
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In addition to the potential therapeutic effects on glucose metabolism, AMPK activation
may also have favorable effects on lipid metabolism. Skeletal muscle insulin resistance under
obesity is associated with elevated intramuscular lipid levels, raising the possibility that
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alterations in lipid metabolism influence insulin signaling (Bonen et al., 2004; Steinberg, 2007).
AMPK activation has been shown to inhibit the key enzymes of lipid and cholesterol
biosynthesis, suggesting the effect of this kinase to ameliorate dyslipidemia (Muoio et al., 1999).
In db/db mice, similar to metformin, treatment with 4a resulted in a dose-dependent decrease in
total cholesterol level in serum and liver. In contrast, serum triglyceride level was significantly
elevated by 4a treatment in db/db mice at higher dose (30 mg/kg). This data conflict the
postulated role of AMPK to decrease lipid synthesis, but chronic AICAR treatment has
previously been reported to increase serum triglycerides in db/db and ob/ob mice (Halseth et al.,
2002). In contrast to the notable increase in serum triglyceride level after 4a treatment, hepatic
and skeletal muscle triglyceride levels were significantly decreased by 4a in db/db mice. This
suggests that increased serum triglycerides level observed upon 4a treatment was not due to
increased production in liver or skeletal muscle. However, the decreased rate of clearance might
be possible reason for the elevated serum triglyceride level upon 4a treatment in db/db mice.
Moreover, treatment with 4a did not cause any significant change in gene expression of ACC,
but there was a significant decrease in Stearoyl-CoA desaturase (SCD1), indicating its beneficial
effect on liver lipid metabolism. SCD1 catalyzes the rate-limiting step in the biosynthesis of
monounsaturated fatty acids and decreased activity of SCD1 facilitates increased energy
expenditure and β-oxidation through activating AMPK (Dobrzyn et al., 2004). Moreover, both
4a treatment reduced the expression of SREBP-1c and PPAR-α, pointing the increase in energy
expenditure and decrease in lipid synthesis in liver.
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AMPK is a principal regulator of energy metabolism and mitochondrial biogenesis (Zong
et al., 2002; Canto et al., 2009). Here, we assessed the effect of 4a-mediated AMPK activation
on body composition and energy balance in db/db mice and found a significant increase in lean
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mass and decrease in fat mass, associated with increase in energy expenditure (in form of heat)
by 4a treatment in db/db mice. The food intake was also higher in 4a treated mice, compared to
control. The fact that energy expenditure was enhanced by 4a treatment throughout the light and
dark cycle, but there was no significant difference in physical activity in control or 4a-treated
mice, suggest that substrate oxidation is continuously higher in 4a-treated mice. In agreement,
4a-treated mice displayed an increased rate of oxygen consumption and carbon dioxide
production, and higher metabolic rates compared to control animals. But there was no significant
difference in RER, reflecting the use of carbohydrates over lipid as a source of energy. Taken
together, these data suggest that treatment with 4a accelerates the substrate metabolism in mice
and hence displays better glucose tolerance, and inhibits further weight gain.
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AMPK has been proposed as a key molecule in eliciting metabolic adaptations to
exercise. Pharmacological and exercise-induced activation of AMPK has been reported to
increase the expression and activity of several mitochondrial genes and oxidative proteins,
including citrate synthase, ATP synthase, cytochrome c oxidase and uncoupling protein 1, and
transcription factors, including PPARα, PGC-1α, NRF1, and Dio2 (Suwa et al., 2003; Terada et
al., 2002). The 4a treatment in db/db mice up regulated the expression of thermogenic markers,
UCP1, PGC-1α, Dio2 and NRF-1 in skeletal muscle of db/db mice, indicating its effect on
energy expenditure through thermogenesis. PGC-1α also acts as a downstream target of AMPK
to modulate energy metabolism (Jager et al., 2007). We observed activation of mitochondrial
genes including ATP synthase, COX IV, and cytochrome C in skeletal muscle of 4a-treated
db/db mice, suggesting its beneficial effect on mitochondrial functions. Collectively, our data
suggested that 4a could activate AMPK that activate PGC-1α leading to the induction
mitochondrial proteins to regulate energy expenditure. In conclusion, our findings establish an
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important role for an isoalantolactone derivative (4a) in regulating skeletal muscle glucose
metabolism and bioenergetics, and provide a basis for further structural activity relationship
(SAR) analysis to develop optimized lead from isoalantolactone for the management of T2DM.
Acknowledgements
The authors gratefully acknowledge the financial support from Indian Council of Medical
Research (ICMR), New Delhi India in the form of ad-hock project (IRIS ID No. 2011-09510).
DA and AKR are also thankful to ICMR for financial support in the form of Senior Research
Fellowship. We would like to thank Dr Amira Klip for providing L6-GLUT4myc cells. This
manuscript bears the CDRI communication No. XXXX.
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References
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