European Journal of Pharmacology 650 (2011) 472–478
Contents lists available at ScienceDirect
European Journal of Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Effect of a novel biphenyl compound, VMNS2e on ob/ob mice
Sucheta B. Kurundkar a,⁎, Narsingh Sachan b, Kisan M. Kodam a,⁎, Vithal M. Kulkarni b,
Subhash L. Bodhankar c, Vikram S. Ghole a
a
b
c
Division of Biochemistry, Department of Chemistry, University of Pune, Pune, India
Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, India
Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, India
a r t i c l e
i n f o
Article history:
Received 14 April 2010
Received in revised form 17 September 2010
Accepted 23 September 2010
Available online 13 October 2010
Keywords:
PTP1B inhibitor
VMNS2e
Anti hyperglycaemic
Insulin sensitizer
Lipid metabolism
(ob/ob mouse)
a b s t r a c t
VMNS2e is a novel biphenyl compound, which in previous studies had showed most favourable interactions
with the active site of protein tyrosine phosphatase 1B (PTP1B). The effect of acute and chronic treatment of
VMNS2e (30 mg/kg) was investigated in ob/ob mice. Plasma glucose was measured after acute administration
of VMNS2e (30 mg/kg) in both lean and ob/ob mice. In the chronic study, VMNS2e (30 mg/kg) was given
orally, once daily for 60 days. Metformin (300 mg/kg) was taken as standard therapy. Body weight, food
intake and blood glucose was measured weekly while glycosylated hemoglobin A1c (HbA1c), insulin,
triglyceride, total cholesterol, low density lipoprotein (LDL), fructosamine, non esterified fatty acid and organ
weight were estimated after the completion of treatment period. Oral glucose tolerance test was performed
on the last day of treatment. Liver and epididymal fat weights were taken. Acute dose of VMNS2e elicited an
anti hyperglycemic effect. It reduced blood glucose by 14% (0.5 h) and 35.6% (6 h). Chronic VMNS2e treatment
improved glucose tolerance by 25.3%. It decreased blood glucose levels. Hyperinsulinemia was reduced
(19.6%). VMNS2e treatment had no significant effect on body weight and food consumption. VMNS2e
treatment exhibited significant reduction (28.2%) in HbA1c, plasma triglyceride (49%), LDL (24%) and
fructosamine (13%) levels. VMNS2e treatment did not alter total cholesterol and non esterified fatty acid
levels. Epididymal fat/body weight ratio was reduced (26.3%). VMNS2e exhibited both acute and chronic anti
hyperglycemic effect, insulin sensitivity along with improvement in various lipid parameters and glycemic
control.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Type 2 diabetes and obesity are characterized by resistance to
hormones (insulin and leptin), possibly due to attenuated or
diminished signaling from the receptors. Pharmacological agents
which are capable of inhibiting the negative regulator(s) of the
signaling pathways are expected to potentiate the action of these
hormones and therefore be beneficial in the treatment of both type 2
diabetes and obesity. A large body of data from cellular, biochemical,
mouse and human genetic and chemical inhibitor studies have
identified protein tyrosine phosphatase 1B (PTP1B) as a major
negative regulator of both insulin and leptin signaling. Coordinated
tyrosine phosphorylation is essential for signaling pathways regulated
by insulin and leptin. In addition, evidence suggests that insulin and
leptin action can be enhanced by the inhibition of PTP1B. As a result of
this, PTP1B has emerged as an attractive novel target for the treatment
of type 2 diabetes and obesity. Link between PTP1B, diabetes and
⁎ Corresponding authors. Tel./fax: + 91 20 25691728.
E-mail addresses: sucheta.kurundkar75@gmail.com (S.B. Kurundkar),
kodam@chem.unipune.ac.in (K.M. Kodam).
0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2010.09.067
obesity has augmented profound increase in research efforts to
explore for new and more PTP1B inhibitors.
Several groups have earlier demonstrated that by employing the
knowledge derived from both structural and medicinal chemistry,
PTP1B inhibitors with the requisite potency and selectivity is now a
viable option to be produced. These findings have strengthened the
discovery and screening of new chemical entity with biphenyl moiety
(Kenner et al., 1996; Malamas et al., 2000; Walchli et al., 2000;
Johnson et al., 2002; Pei et al., 2004; Sachan et al., 2007; Sachan et al.,
2009). Now the challenge for the future will be to transform this
potent and selective small molecule (PTP1B inhibitor) into orally
available drugs with desirable physicochemical properties and in vivo
efficacies (Zhang and Lee, 2003). Many treatments are available for
diabetes, including sulfonylureas, thiazolidinediones, biguanides and
α-glycosidase inhibitors. However, the growing number of diabetic
patients highlights the need for new treatments. Current treatment
options are often effective over the short and medium term, but do
not alter the underlying progression of the disease. In addition, weight
gain, hypoglycemia, gastrointestinal events and peripheral edema are
important adverse events associated with these agents which limit
compliance. The need for new treatments is widely recognized and
the search is on (Cole et al., 2008).
S.B. Kurundkar et al. / European Journal of Pharmacology 650 (2011) 472–478
Among all synthesized biphenyl compounds in our laboratory,
VMNS2e showed most favourable interactions with the active site of
PTP1B by docking and molecular dynamics (Sachan et al., 2009) so we
had selected this compound for this present study. VMNS2e was
found to possess potent in vivo hypoglycemic activity and improved
glucose tolerance property when administered in alloxan induced
diabetic mice (Sachan et al., 2009). We had selected ob/ob mice as
they are hyperphagic, obese, hyperinsulinemic, hyperglycaemic and
they are used as a model for diabetes and obesity (Lindström, 2007).
The objective of the present study was to evaluate the effects of a
novel biphenyl compound, VMNS2e (C20H16NO4) (1-biphenyl-4-yl-2(4-nitro-phenoxy)-ethanone) (Fig. 1) in ob/ob mice. In this study, we
evaluated the effects of VMNS2e (30 mg/kg) on glucose tolerance,
insulin, glycosylated haemoglobin levels, as well as on body weight,
food intake and various lipid parameters and organ weights.
2. Materials and methods
2.1. Animals and research protocol approval
Male obese diabetic mice (ob/ob) with C57BL/6 J background (B6.
VLepob(−/−)) of 10–12 weeks (Reul et al., 1997) were obtained from
Jackson Laboratory, Bar Harbor (Maine, USA) (Xabier et al., 2000).
Lean male mice (+/+) with C57BL/6 J background of 10 to 12 weeks
were obtained from Raj Biotech (India) Pvt. Ltd, Pune. Mice were
housed in groups in individually ventilated cages under standard
conditions with 12/12 h light-dark cycles, temperature (23 ± 2 °C),
relative humidity (55 ± 5%). Ob/ob mice were fed with standard
rodent chow (5 K20 irradiated feed, Lab diet, U.S.A.) 5 K20 pelleted
food contained minimum of 10% fat needed to meet the metabolic
requirements of ob/ob mice. Lean mice were fed with standard rodent
chow (Chakhan, Nav Maharashtra, India). Autoclaved rice husk was
used as bedding and UV treated water was given. Both food and water
were given ad libitum. In acute study food was withdrawn during the
study period but water was provided ad libitum. Animal handling was
performed as per Good Laboratory Practice and as per applicable
national/international guidelines. The Institutional Animal Care and
Use Ethics Committee (IAEC) under The Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA)
approved the research proposal (approval no.24/2006-07) and
conformed to European Community guidelines. Unless otherwise
stated, all were of reagent-grade quality.
2.2. Drug preparation and administration
VMNS2e was synthesized in our laboratory as per method
described by Sachan et al. (2009). VMNS2e was suspended in vehicle
O
O
NO2
Fig. 1. Chemical structure of VMNS2e (1-Biphenyl-4-yl-2-(4-nitro-phenoxy)-ethanone
(Molecular weight-334.35).
473
comprising of 0.5% w/v sodium carboxy methyl cellulose (Qualigens)
and 2.0% Tween 80 (Qualigens). Metformin (1,1-dimethylbiguanide
hydrochloride, USV Ltd., India) was dissolved in water. Both VMNS2e
and metformin were orally administered using an oral gavage tube
once daily. High pressure liquid chromatography studies have shown
that the compound VMNS2e was 97% pure.
2.3. Acute treatment study
Male ob/ob mice with blood glucose level above 16.7 mmol/l in the
fed state were randomized into 2 groups (n= 5). A single oral dose of
VMNS2e (30 mg/kg) in vehicle was administered orally by gavage tube
while control group received only vehicle. Lean male C57BL/6 J mice was
also randomly divided into similar 2 groups as described above (n= 5).
Blood was collected from retro orbital plexus (20–80 μl) under light
ether anaesthesia (Anaesthetic ether, Qualigens, India) and plasma
glucose was measured at 0, 0.5, 2, 4, 6 and 18 h after oral dosing by
glucose oxidase method (Martins et al., 2002). Percentage reduction in
blood glucose was calculated with respect to control group. A 10%
reduction in blood glucose level versus control group was considered as
a positive screening result (Hodge et al., 2010).
2.4. Long term treatment study
Male ob/ob mice with fasting blood glucose level above
13.9 mmol/l were selected for the study. The ob/ob mice were randomly
assigned to 3 groups based on fasting blood glucose values (first
criterion) and initial body weight (second criterion) (Hu et al., 2006)
(n= 6): obese-diabetic control (control) group and two treatment
groups. The control mice were treated with vehicle only. VMNS2e was
orally administered daily once for 60 days in vehicle at the dose of
30 mg/kg. Metformin was used as a standard treatment and administered at a dose of 300 mg/kg (Cohen et al., 2004) once daily for 60 days.
During the 60 days treatment period body weight, food intake and blood
glucose were measured weekly. At the end of the study, blood (500–
1000 μl) was collected from retro orbital plexus under light ether
anaesthesia for determination of various biochemical parameters. The
mice were euthanized using carbon dioxide asphyxiation method. The
organs were carefully dissected out and weighed.
2.4.1. Body weight and food intake
Body weights and food consumption were measured weekly using
a calibrated weighing balance (Citizen, India) by placing mice in
metabolic cages (Techniplast, Italy).
2.4.2. Oral glucose tolerance test
Oral glucose tolerance test was performed at the end of the
treatment period (Reul et al., 1997). Animals were fasted overnight
(10 to 12 h) and next day glucose load (1.5 g/kg, Qualigens) was orally
administered (Kubota et al., 2006). Plasma glucose levels were
measured at 0 (before glucose challenge), 30, 60, and 120 min after
glucose load by glucose oxidase method using commercial kit (Merck)
(Martins et al., 2002). The area under the curve (AUC) for glucose was
calculated as per Hu et al. (2006). The AUC was used as a measure of
glucose tolerance.
2.4.3. Plasma insulin
After the completion of treatment, plasma insulin was measured
according to the method described by Gum et al. (2003) using mouse
insulin ELISA kit (ALPCO Diagnostics, USA).
2.4.4. Glycosylated haemoglobin levels
The percentage of glycosylated hemoglobin A1c (HbA1c) was
measured by high performance liquid chromatography (Bio-Rad D-10,
Hemoglobin Testing System, U.S.A.) (Guorong et al., 2004) after
completion of the treatment period.
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2.4.5. Lipid parameters
Plasma triglyceride and total cholesterol levels were measured
using colorimetric enzymatic assay method (GPO and CHOD/PAP
method respectively) using commercial kit (Teco diagnostics and
Crest biosystem respectively) (Martins et al., 2002). Direct low
density lipoprotein (LDL) was measured using commercial kit (Teco
diagnostics). Non esterified fatty acid concentrations were assayed
using commercial kit (Randox Laboratories, UK) (Reul et al., 1997;
Roche et al., 2002). Early glycation product, fructosamine was
determined by standard clinical laboratory procedure using commercial kit (Roche Diagnostics GmbH, Mannheim) as described by Nardai
et al. (2005) in Hitachi 912 Clinical Chemistry analyzer (Roche
Diagnostics GmbH, Mannheim).
2.4.6. Liver and epididymal fat
Weight of liver and epididymal fat were measured in calibrated
electronic weighing balance (AX 120, Shimadzu, Japan). The relative
organ weight (%) was calculated based on final body weight (Lin et al.,
2000; Hue et al., 2009).
2.5. Statistical analysis
Data were expressed as mean± S.E.M. and analyzed using one-way
ANOVA followed by post hoc comparisons test (Dunnett's test). P-value
less than 0.05 were considered significant. Data were analyzed
statistically using Graph Pad Software (Instat version 3.0, U.S.A.).
3. Results
3.1. Effect of VMNS2e after acute treatment in ob/ob mice
Acute administration of VMNS2e (30 mg/kg) in ob/ob mice
resulted in 14% reduction in blood glucose level at 0.5 h which was
considered as positive screening result. Percentage of blood glucose
reduction was found to be maximal at 6 h (35.6%) after VMNS2e
administration. The results thus show a quick onset (0.5 h) of anti
hyperglycemic action and significant effect at 4–6 h while its peak
effect was at 6 h (Table 1A). Acute oral administration of VMNS2e
(30 mg/kg) to normal non-diabetic mice did not significantly alter
plasma glucose levels (data not shown).
3.2. Effect of VMNS2e after chronic treatment in ob/ob mice
3.2.1. Effect of VMNS2e on blood glucose level
Chronic administration of VMNS2e (30 mg/kg) daily for 60 days
reduced the mean plasma glucose concentration from 24.84 to
11.48 mmol/l. The plasma glucose level was significantly lowered
after 30 days of treatment and its effect was maintained till 60th day.
The effect of reduction was increased (P b 0.01) on 60th day as
compared to before treatment. Metformin showed significant decrease from day 21 and it reduced blood glucose more effectively
(P b 0.01) from day 53. The reduction in the fasting blood glucose
levels was earlier in time and more evident in metformin as compared
to VMNS2e treated ob/ob mice but VMNS2e showed consistent
performance in reduction of blood glucose as compared to metformin
(Fig. 2).
Table 1A
Effect of acute oral treatment of VMNS2e (30 mg/kg) on blood glucose % reduction in
ob/ob mice.
Baseline
(0 h)
0.5 h
2h
4h
6h
18 h
0.2 ± 1.6
−14.0 ± 6.1
− 11.5 ± 11.3
− 29.9 ± 4.5a
− 35.6 ± 3.9a
− 7.1 ± 4.0
Mean ± S.E.M. One way ANOVA followed by Dunnett's multiple comparison. n = 5.
a
P b 0.01 (when compared with baseline).
Fig. 2. Effects of VMNS2e on fasting blood glucose level in ob/ob mice. n = 6. Mean± S.E.M.
*Pb 0.05, ** P b 0.01 Vs before treatment and #P b 0.05, ##Pb 0.01 Vs vehicle treated mice.
3.2.2. Effect of VMNS2e on body weight and food intake
In vehicle treated ob/ob mice, a trend of increase in body weight
was observed and the net gain in body weight was 8 g during the
60 days treatment period. VMNS2e treatment resulted in less increase
in body weight (6 g). On the other hand, metformin treatment
resulted in marginal increase in body weight, a meagre gain of 0.83 g
compared to that of day 0 (before treatment) (data not shown).
Food intake of control mice was uniform throughout the treatment
period (data not shown). Metformin treatment caused significant
reduction in food consumption (34%) whereas VMNS2e treatment
had no significant effect on food consumption (8%). Reduction in the
body weight by metformin treatment may be strongly associated with
the decrease in food consumption.
3.2.3. Effect of VMNS2e on oral glucose tolerance test
The AUC calculated after oral glucose load in vehicle treated control
mice was 16.6 mmol/l/min. Both metformin (10.7 mmol/l/min) and
VMNS2e (12.4 mmol/l/min) treatment showed significant reduction in
AUCglucose by 35.5 and 25.3% respectively compared with the control AUC
(vehicle treated ob/ob mice). Metformin treatment seemed to be more
effective in reducing the AUCglucose compared to VMNS2e, although the
difference was not significant. The result thus indicates that VMNS2e
treatment increased utilization of glucose and improved glucose
tolerance in ob/ob mice after long term treatment (Fig. 3A and B).
3.2.4. Effect of VMNS2e on plasma insulin levels
Ob/ob mice exhibited marked hyperinsulinemia. Metformin
treatment reduced the plasma insulin (pmol/l) levels significantly
(P b 0.05) from 30.9 ± 2.1 to 25.3 ± 1.0, while VMNS2e treatment
reduced (P b 0.05) it to 24.8 ± 0.8. VMNS2e treatment reduced (19.6%)
plasma insulin slightly more than that reduced by metformin (18%).
Both metformin and VMNS2e treatment showed decrease in
circulating insulin levels in obese mice. The results thus indicate
that the effect of both the drugs results in the improvement of insulin
action by positively enhancing the insulin signalling pathway.
3.2.5. Effect of VMNS2e on glycated hemoglobin (HbA1c) levels
HbA1c levels in the metformin as well as VMNS2e (30 mg/kg)
treated mice were lower than that in the control group. HbA1c level in
control group was 6.5 ± 0.5. The reduction in HbA1c level was more in
VMNS2e treated (4.7 ± 0.4) (28%) group than in metformin treated
S.B. Kurundkar et al. / European Journal of Pharmacology 650 (2011) 472–478
475
(30 mg/kg) remarkably decreased hyperlipidemia by reducing the lipid
levels in the ob/ob mice, when treated for 60 days as compared to obese
control. It is interesting to note that both metformin and VMNS2e
reduced LDL cholesterol and fructosamine levels, while they behaved
differently in reduction of other lipid parameters (plasma triglycerides
and total cholesterol levels).
3.2.7. Effect of VMNS2e on liver and epididymal fat weight
VMNS2e treatment in ob/ob mice for 60 days resulted in significant
reduction (26.3%) in the epididymal fat/body weight ratio compared
with the control while it increased the liver/body weight ratio by
38.3%. Metformin treatment did not alter epididymal fat/body weight
ratio in ob/ob mice post 60 days of treatment; it significantly reduced
liver/body weight ratio by 34.5% (Table 1B).
4. Discussion
Fig. 3. Effects of VMNS2e on oral glucose tolerance test in ob/ob mice. Ob/ob mice were
treated with VMNS2e (30 mg/kg), metformin (300 mg/kg) and vehicle (obese mice
treated with vehicle) for 60 days and oral glucose tolerance test was performed on last day
of treatment. (A) Blood glucose levels during oral glucose tolerance test, and *Pb 0.05,
** P b 0.01 compared with vehicle and ##P b 0.01 compared with time 0 min. (B) AUC (area
under curve) of oral glucose tolerance test. n = 6. Mean± S.E.M. *Pb 0.05, ** P b 0.01
compared with vehicle and ##P b 0.01 compared with metformin.
(5.6 ± 0.4) group (13.8%). The results thus indicated that VMNS2e
exhibited improved glycaemic control in ob/ob mice.
3.2.6. Effect of lipid profile
Plasma triglyceride (mg/dl) level in control group of mice was
186.2 ± 7.8. Metformin treatment was ineffective in lowering the
triglyceride level whereas VMNS2e treatment significantly reduced
(49.2%) the triglyceride level (Fig. 4A). On the other hand, metformin
significantly reduced (30.4%) total cholesterol compared to control
mice. VMNS2e treatment reduced (12%) total cholesterol levels in ob/ob
mice however this reduction was insignificant (Fig. 4B). Metformin
treatment reduced LDL level by 41.2% while VMNS2e reduced it by 24%
(Fig. 4C). Both metformin and VMNS2e treatment reduced plasma
fructosamine level by 22.6% and 13% respectively (Fig. 4D). Non
esterified fatty acid levels were unaffected by both treatments. The
result thus indicates that both metformin (300 mg/kg) and VMNS2e
Ob/ob mice were used in this present study because they have a
defect in the gene for leptin, a protein involved in appetite regulation
and energy metabolism (Haluzik et al., 2004) and are hyperphagic,
obese and insulin resistant (Lin et al., 2000), hence serving as a good
animal model for the evaluation of anti hyperglycemic and insulinsensitizing drugs. Ob/ob mice also have an unique lipoprotein referred to
as low-density lipoprotein, LDL (Plummer and Hasty, 2008). They also
have fatty liver leading to hepatic steatosis (Margalit et al., 2006).
Sachan et al (2009) reported LD50 of VMNS2e to be above 2000 mg/kg in
mice. The dose of VMNS2e was chosen on the basis of previous studies
conducted in our laboratory (Kurundkar et al., in press) and reported
results (Sachan et al., 2009). We have studied VMNS2e 60 mg/kg as well
as 120 mg/kg in other animal models and ascertained its efficacy and
safety. VMNS2e showed anti diabetic and renoprotective activity in 30
and 60 mg/kg body weight in STZ induced long-term diabetic rats.
However, in higher dose of 120 mg/kg it did not exhibit dose dependent
action. The effect of VMNS2e at various dose levels of 30, 60 and 120 mg/
kg on various biochemical, haematological, physiological, behavioural
and histological parameters were studied (unpublished data). Results
obtained from this study showed that VMNS2e exerted an acute and
chronically sustained anti hyperglycaemic effect. The ability of VMNS2e
to reduce hyperglycaemia can be attributed to its insulin sensitizing
effect. This insulin sensitizing and anti-diabetic activity of VMNS2e
indicates PTP1B inhibition as the possible mechanism responsible for
the observed effect. Change in food consumption cannot be attributed as
food was withheld during acute study and was not significantly altered
during chronic VMNS2e treatment. Decrease of marked hyperinsulinemia was evident as a result of chronic treatment by both VMNS2e and
metformin. This does not preclude the possibility that VMNS2e might be
improving the insulin action by positively enhancing the insulin
signalling pathway. VMNS2e differed from metformin in many ways
like metformin was more effective than VMNS2e in reducing fasting
blood glucose levels. Metformin reduced body weight more than
VMNS2e and induced anorexia which was not seen in VMNS2e treated
mice. There was also difference in its effect on the triglyceride and total
cholesterol level and relative liver and epididymal fat weight. Reduction
in the rate of glucose production by metformin is due to reduction in
gluconeogenesis (Hundal et al., 2000). The mechanism by which
VMNS2e acts is unknown and needs to be studied in greater detail.
Impaired glucose tolerance observed in the control mice confirmed the insulin resistant state of ob/ob mice (Hu et al., 2006).
Improvement in glucose tolerance after VMNS2e administration
suggests that VMNS2e could possibly improve insulin resistance
state and may cause an increase in the whole body insulin sensitivity
in severely insulin resistant ob/ob mice (Bailey and Flatt, 1997).
Reduced metabolic rate contributed to weight gain in leptin-deficient
(ob/ob) mouse (Breslow et al., 1999). This is in line with our study
findings wherein the control mice exhibited weight gain. Cheah
(1998) reported that weight loss has a significant co-relation in the
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Fig. 4. Effect of VMNS2e (30 mg/kg) and metformin (300 mg/kg) treatment on lipid levels in ob/ob mice after 60 days of treatment. Triglyceride (A), Total cholesterol (B), Low
Density Lipoprotein (C), and Fructosamine (D). n = 6. Mean ± S.E.M. *P b 0.05, **P b 0.01 compared to vehicle.
improvement of glucose tolerance, insulin sensitivity. It reduces both
lipid levels and blood pressure. Kim et al. (2006) reported that
metformin treatment had enhancing effect on leptin's anorexic
activity and body weight–losing effects in standard chow rats. They
also reported similar effect of metformin in high fat fed obese rats.
Various researchers (Lin et al., 2000; Wong and Wong, 2003) have
already reported that metformin leads to anorexia and weight loss.
This weight loss is however not desired in elderly patients with
NIDDM (Wong and Wong, 2003). We observed that metformin
treated ob/ob mice showed decrease in food intake thereby registering
reduction in body weight gain. VMNS2e did not exhibit anorectic
behaviour as observed in metformin group. Earlier study by Sachan et
al. (2009) showed increase in body weight by VMNS2e treatment in
diabetic mice, whereas VMNS2e treatment in leptin deficient and
insulin resistant ob/ob mice did not exhibit this pattern. The
mechanism by which this observation differs is not understood
clearly and calls for a detailed investigation and additional study.
Glycated haemoglobin (glucose memory test) of control mice was
increased in control group. Rains et al. (1989) reported reduction in
HbA1c levels by the metformin therapy. We found that VMNS2e was
slightly more effective than metformin in reducing the HbA1c levels.
This observation would be of clinical significance as there are very few
available drugs which show a specific effect on glycated haemoglobin.
Table 1B
Effect of VMNS2e (30 mg/kg) on relative organ weight % in ob/ob mice.
Groups
Liver/body weight
(%)
Epididymal/body weight
(%)
OC
MT300
VMNS2e 30
7.62 ± 0.15
4.99 ± 0.26a
10.54 ± 0.35a
3.65 ± 0.26
3.17 ± 0.20
2.69 ± 0.10a
MT300 — Metformin (300 mg/kg); VMNS2e 30 — VMNS2e (30 mg/kg) Mean ± S.E.M.
One way ANOVA followed by Dunnett's multiple comparison. n = 6.
a
P b 0.01 (when compared with vehicle).
This is in contrast to the findings observed by Chyan and Chuang
(2007), where they reported that long term metformin treatment for
52 weeks resulted in deterioration of glycemic control indicating
increase in the HbA1c levels in type 2 diabetics. It is interesting to note
that reduction offered by VMNS2e treatment, maintained the plasma
glucose level in the chronic study and the effect was found to be more
consistent as compared to the metformin group from day 30 to day 60,
this may in turn contribute to this finding, indicating that only long
term, continuous treatment (after 30 day at least) may induce
sustained anti hyperglycaemic effect and contribute in improving
the glycaemic control. This consistent performance of VMNS2e over
metformin was however found to be not significant.
Metabolic profile of type 2 diabetes showed impaired glucose
metabolism, insulin resistance frequently combined with dyslipidemia
(Cha et al., 2005). Ob/ob mice also exhibit similar condition. VMNS2e
reduced plasma triglyceride levels whereas total cholesterol levels
remain unaffected. This is in contrast with that of metformin treatment
wherein total cholesterol was reduced but plasma triglyceride levels
were unaffected. Metformin findings were similar to those observed by
Pentikäinen et al. (1990) and Wulffele et al. (2002). In vitro study by
Patanè et al. (2000) showed that metformin decreased the elevated
triglyceride content of islets cultured in the presence of free fatty acid. In
Zucker diabetic fatty rat, addition of metformin to food showed delayed
onset of diabetes, decreased islet triglyceride levels, elevated basal
insulin secretion and increased the ability of β-cells to respond to
glucose stimulation (Sreenan et al., 1996). In insulin-resistant condition,
non esterified fatty acid production is increased due to lipolysis in the
peripheral adipose tissue. Many tissues (liver and skeletal muscle) when
exposed to high free fatty acid level can cause an increase in insulin
resistance. So an increase in non esterified fatty acid concentration is
reported for insulin resistance. No difference in non esterified fatty acid
levels following treatment with either metformin or VMNS2e was
observed compared with the control group as reported earlier (James
et al., 2005). This may be possibly due to its improvement in insulin
sensitivity by both the treatments. Ida et al. (2003) reported that
S.B. Kurundkar et al. / European Journal of Pharmacology 650 (2011) 472–478
metformin when given for 4 weeks in Goto–Kakizaki rat had no effect on
non esterified fatty acid levels.
Formation of fructosamine increases with the level of blood glucose.
Its metabolism occurs within 1 to 3 weeks, corresponding to the
turnover of most of the serum proteins. Fructosamine concentration
thus reflects average of the continuously varying blood glucose
concentrations during this period, serving as a blood glucose memory.
Fructosamine is therefore a rapid indicator of glycaemia in the diagnosis
and management of diabetes mellitus (Johnson et al., 1983; Armbruster,
1987; Henrichs, 1990; Cefalu et al., 1991). Both metformin and VMNS2e
treatment reduced the circulating fructosamine levels in ob/ob mice.
This is similar to the findings reported by Garber et al. (2003) wherein
they had stated that metformin reduced fructosamine levels in type 2
diabetics. Schwartz et al. (2006) also stated the same but added that this
reduction in fructosamine level is better when given in extendedrelease form. This finding can be co-related with the improvement in the
glycaemic control by reducing the HbA1c levels.
LDL is already recognized as the key factor in the pathogenesis of
artherosclerosis and coronary artery disease. NIDDM subjects on a long
term metformin therapy (6 weeks) showed reduction in of LDL levels
(Rains et al., 1989). This is in line with our study findings. VMNS2e also
reduced LDL level which was at par with that of metformin therapy.
Pentikäinen et al. (1990) reported that 9 weeks of metformin treatment
reduced total cholesterol and LDL levels (Pentikäinen et al., 1990).
DeFronzo and his group (1995) reported that after 29 weeks of
metformin treatment both triglyceride and LDL cholesterol level were
decreased. The difference between the animal model (ob/ob mice) with
obese subjects showed larger differences and this can be attributed to
difference in the treatment time along with species, while we had
administered metformin for 60 days (approximately 8 weeks), DeFronzo
and his group had administered for much longer period of 29 weeks.
Metformin treatment had no effect on the relative epididymal fat weight
(normalized to body weight) as reported by Lin et al. (2000). Similar
findings were also observed in rats (Suwa et al., 2006). Relative liver
weight was increased with VMNS2e treatment. It is difficult to
comprehend the reason behind this finding. More elaborate study is
warranted to understand the reason which might contribute to the
increase in relative liver weight even when both plasma triglyceride and
LDL levels were considerably reduced. Liver lipid content analysis may be
able to address whether there was any significant alterations in the lipid
levels in the liver upon treatment with metformin and VMNS2e,
particularly in the light of the fact that VMNS2e increased liver weight
and reduced epididymal fat and this is being addressed in our
forthcoming study.
VMNS2e treatment significantly reduced relative epididymal fat
weight and this finding is very similar to those observed by Zinker et al.
(2002). This might possibly be due to alteration in the lipid metabolism
(Zinker et al., 2002). Reduction in liver index (liver/body weight %) in
fatty rats after 4 weeks of metformin treatment was reported earlier
(Gao et al., 2005). It is difficult to comment if the reduction of
epididymal fat may contribute to hepatic fat storage. However, Zinker
et al. (2002) also reported similar findings when they treated PTP1b
antisense oligonucleotide in ob/ob mice. We need to evaluate whether it
is a typical rodent-specific effect or whether it has any other relationship
between PTP1B inhibitor and epididymal fat.
Zinker et al. (2002) screened a PTP1B antisense oligonucleotide in
ob/ob mice for 42 days (6 weeks) and reported that the treatment
normalized plasma glucose levels, postprandial glucose excursion, and
HbA1C. PTP1B protein and mRNA were reduced in liver and fat with no
effect in skeletal muscle. We used a biphenyl compound, PTP1B
inhibitor for 60 days treatment in ob/ob mice. We also witnessed
similar findings like Zinker et al. (2002) wherein the hyperinsulinemia
was reduced due to improved insulin sensitivity and blood glucose
levels were reduced. We also observed similar trend in the weight of
liver and epididymal fat in ob/ob mice when treated with PTP1B
antisense (25 mg/kg). More significant effect was observed by PTP1B
477
antisense treatment in the oral glucose tolerance study compared to
VMNS2e. Additionally, we observed that VMNS2e lowered plasma
triglycerides, low density lipoprotein and fructosamine in ob/ob mice.
We however did not study PTP1B protein and mRNA levels while Zinker
and his colleagues did not study the lipid profile.
VMNS2e showed anti diabetic and renoprotective activity in 30
and 60 mg/kg body weight in STZ induced long-term diabetic rats
(Kurundkar et al., in press). However, in higher dose of 120 mg/kg it
did not exhibit dose dependent action. The effect of VMNS2e at
various dose levels of 30, 60 and 120 mg/kg on various biochemical,
haematological, physiological and histological parameters are studied
(unpublished data).
5. Conclusion
In conclusion this study had demonstrated that VMNS2e exerts an
acute and chronic anti hyperglycemic effect in ob/ob mice. PTP1b
inhibition might be the mechanism responsible for the observed effect.
VMNS2e lowered plasma triglycerides, low density lipoprotein and
fructosamine in peripheral circulation. However, with the exception of
triglyceride, HbA1c levels and epididymal fat, all the parameters seem to
improve better with metformin.
Acknowledgements
Authors are thankful to Raj Biotech (India) Pvt. Ltd. for providing all
the facilities to carry out the animal studies. Authors thank Dr. Vasant V.
Joshi (USV Ltd., India) for providing gift sample of metformin.
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