Rajesh Kumar Suman et al / International Journal of Biomedical Research 2016; 7(2): 055-065.

55
International Journal of Biomedical Research
ISSN: 0976-9633 (Online); 2455-0566 (Print)
Journal DOI: 10.7439/ijbr
CODEN: IJBRFA Original Research Article

Metformin ameliorates diabetes with metabolic syndrome induced changes in

experimental rats

Rajesh Kumar Suman1, Ipseeta Ray Mohanty1, Ujawala Maheshwari2, Manjusha K. Borde1 and
Y A Deshmukh1
1Department of Pharmacology, MGM Medical College, Sec-01, Kamothe, Navi Mumbai-410209
2Department of Pathology, MGM Medical College, Sec-01, Kamothe, Navi Mumbai-410209

*Correspondence Info:
Dr. Rajesh Kumar Suman,
Tutor, Dept. of Pharmacology, MGM Medical College
Sec-01, Kamothe-410209, Navi Mumbai
E-mail: rajeshsuman2043@gmail.com

Abstract
Objective: Metformin is the primary glucose- lowering agent used in the treatment of type II diabetes mellitus. However, there
is no experimental evidence presently available with regard to the possible beneficial effects of Metformin on metabolic
syndrome co-existing with diabetes in experimental rats. Thus, the present study was designed to evaluate potential effects of
Metformin on various components of metabolic syndrome. Also to elucidate the underlying mechanisms: DPP-IV, anti-
inflammatory, antioxidant pathways.
Material and Methods: A combination of high fat diet (HFD) and low dose of streptozotocin (STZ) 40 mg/kg was used to
induce metabolic syndrome co-existing with diabetes mellitus in Wistar rats. The HFD were fed to rats for 10 weeks to induce
metabolic syndrome. At the end of 3 weeks, diabetes was induced by a single STZ injection (40 mg/kg body wt). Metformin
(100 mg/kg) was administered to rat from 4th to 10th weeks daily and various parameters of Diabetes and metabolic syndrome
were studied. Also to understand the mechanisms; DPP-IV pathway, anti-inflammatory, antioxidant parameters were studied.
Biochemical indices of injury {pancreatic, liver and renal function} and histopathological assessment of injury was evaluated
in experimental groups.
Results: Metformin treatment ameliorated the deleterious effects associated with metabolic syndrome and diabetes. Metformin
favorably modulated various parameters: anti-diabetic (reduced Blood glucose, HbA1c, HOMA-IR, increased serum insulin,
HOMA-β and restoration of pancreatic function), Central obesity (reduced body weight, abdominal circumference (AC),
thoracic circumference (TC), AC/TC ratio) and hypolipidemic (favorable lipid profile, artherogenic index). In addition
significant restoration of cardiac injury as indicated by CPK-MB levels were observed. In addition, DPP-IV pathway (reduced
serum DPP-IV), anti-inflammatory (reduced hs-CRP levels), antioxidant (reduced MDA) contributed to the beneficial effects
of Metformin in diabetes with metabolic syndrome. The histopathological assessment confirmed the protective effects of
Metformin on heart, thoracic aorta, pancreas, liver and kidney.
Conclusion: Metformin treatment ameliorated the deleterious effects associated metabolic syndrome in the setting of diabetes
mellitus. Beneficial effects of Metformin can be attributed to hypoglycemic, hypolipidemic, antioxidant, cardioprotective and
anti-inflammatory effects.
Keywords: Metformin, Diabetes, Metabolic Syndrome, High Fat Diet, Streptozotocin.
1. Introduction
The metabolic syndrome represents a cluster of Metformin an insulin-sensitizing biguanide, exerts
abnormalities, including obesity, insulin resistance, an anti-hyperglycemic effect, with minimal risk of
dyslipidaemia and type II diabetes, which increases the risk of hypoglycemia, and has been used to prevent type II diabetes
developing cardiovascular diseases. The Metabolic with a 31% reduction in incidence [2]. The anti-diabetic effect
Syndrome is a clinically and socially important issue which of Metformin owes to its ability to suppress hepatic glucose
has drawn the attention of many physicians and researchers. production, enhance peripheral glucose uptake and improve
Studies have demonstrated that patients with metabolic peripheral insulin sensitivity [3-5]. In fact, Metformin showed
syndrome displayed an increased risk of developing diabetes, beneficial effects in type II diabetes, including weight
cardiovascular disease and other diseases [1]. Therefore the reduction, improved lipid profiles, and enhanced endothelial
co-existence of diabetes with metabolic syndrome is a unique function. There are evidences for a potential role of
pathology that needs to be addressed as a separate entity. Metformin in the prevention of type II diabetes in obese
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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 56
patients [6]. Several studies have suggested that Metformin Ethics Committee and conforms to the Committee for the
may improve some of the features of the metabolic syndrome Purpose of Control and Supervision of Experiments on
as it not only improves insulin sensitivity in the liver and Animals and Indian National Science Academy and
muscle, as its primary anti-hyperglycemic mechanism of Guidelines for the Use and Care of Experimental Animals in
action, but also induces additional beneficial effects on Research. Rats were kept in polyacrylic cages (38×23×15 cm)
several metabolic abnormalities associated with the metabolic with not more than four animals per cage and housed in an
syndrome [7-9]. Recent studies also reported that Metformin air-conditioned room, kept under natural light-dark cycles.
induced improvement of metabolic disorders is associated The animals were allowed free access to standard diet or high
with the energy state of the body, reduction of macrovascular fat diet as the case may be and water ad libitum.
morbidity and mortality, anti-artherogenic, anti-inflammatory 2.3 Preparation High Fat Diet
and antioxidant effects [10]. In combination with lifestyle The High Fat Diet (HFD) was prepared indigenously
modifications, Metformin is the primary glucose- lowering in our laboratory by using Normal Pellet Diet, Raw
agent used in the treatment of type II diabetes mellitus, due to Cholesterol, Mixture of Vanaspati ghee and Coconut oil (2:1).
its efficacy, safety and beneficial cardiovascular and meta- Normal Rat Pellet diet was powdered by grinding and mixed
bolic activity. However, there is no experimental evidence with 2.5% Cholesterol and Mixture of Vanaspati ghee and
presently available with regard to the mitigating effects of Coconut oil (5%). The mixture was made into pellet form and
Metformin on diabetes with metabolic syndrome (HFD-STZ put into freezer to solidify. In addition 2% raw cholesterol
induced). Also the potential of Metformin to modulate the powder was mixed in coconut oil and administered to the rats
DPP-IV Pathway has not been studied so far. The DPP-IV is by oral route (3 ml/kg) [12].
novel adipokine and to characterize the association of DPP-IV 2.4 Experimental model of diabetes with metabolic
to different parameters of metabolic syndrome and that may syndrome
impair insulin sensitivity in an autocrine and paracrine The High Fat Diet (HFD) along with 2% liquid
fashion, DPP-IV release strongly correlates with adipocyte cholesterol (3 ml/kg) was orally fed to rats for 3 weeks to
size, potentially representing an important source of DPP-IV induce metabolic syndrome. After 3 weeks of dietary
in obesity linking with metabolic syndrome[11]. manipulation, overnight fasted rats were injected
The present study was designed to evaluate potential intraperitoneally with STZ (40 mg /kg) [12]. The animals
effects of Metformin on various components of metabolic were allowed to drink 5% glucose solution overnight to
syndrome (Blood glucose, HbA1c, Seum insulin, HOMA- IR, overcome drug induced hypoglycemia. The body weight and
HOMA- β, C-peptide), Central obesity (Body weight, biochemical parameters (Blood glucose, total cholesterol)
abdominal circumference (AC), Thoracic circumference were estimated 7 days after the vehicle or STZ injection, i.e.,
(TC), AC/TC ratio) and dyslipidemic (lipid profile, on 4 weeks of dietary manipulation in rats. The rats with
artherogenic index) with diabetes as an essential component. blood glucose (>200 mg/dl), Total Cholesterol (>110 mg/dl),
Also to understand the underlying mechanisms: DPP-IV triglyceride (>150 mg/dl), change in body weight (8% of
pathway (serum DPP-IV), anti-inflammatory (hs-CRP), initial weight), Systolic Blood pressure (>130 mm/hg) and
antioxidant (MDA), cardioprotective activities (CPK-MB), reduced HDL levels (<35mg/dl) confirmed presence of
safety parameters {pancreatic function [lipase (U/L)], liver metabolic syndrome with diabetes. Thereafter the rats were
function [SGPT (U/L)], renal function [creatinine (mg/dl)]} either fed normal diet or HFD as per the protocol for 10
and histopathological indices of injury in the experimental weeks. Blood samples were collected from the retro-orbital
model of diabetes with metabolic syndrome were studied. plexus under light anesthesia at 0, 4, 7 and 10 weeks for
estimation of biochemical parameters. At the ends of
2. Material and Methods experimental period, rats were sacrificed for
2.1 Chemicals and drugs: histtopathological evaluation of injury to the Heart, Thoracic
Streptozotocin (STZ) was procured from Sigma aorta, Pancreas, Liver and Kidney.
Chemicals St Louis, USA. Cholesterol was procured from 2.5 Experimental Groups
Alfa Aesar and the test drug Metformin was obtained gift Group 1: Normal Control (NC): In Normal Control group,
sample from Cipla pharmaceuticals. All other chemicals and Rats were administered distilled water per orally using a
reagents used were of analytical grade. feeding cannula for study period 10 weeks. At the end of 3
2.2 Experimental Animal week, 0.01 M citrate buffer, pH 4.5 was injected
Adult male Wistar rats, 10 to 12 weeks old, intraperitoneally to mimic the STZ injections.
weighing 150 to 200 gm were used in the study. The rats were Group 2: High Fat Diabetic Control (HF-DC): The HFD
housed in the Central Animal Facility of our own MGM was fed to rats for 10 weeks to produce metabolic syndrome.
Medical College, Navi Mumbai, India. They were maintained At the end of 3 week diabetes was induced by a single STZ
under standard laboratory conditions in the animal house. The injection (40 mg/kg body wt, i.p. dissolved in 0.01 M citrate
study protocol was approved by the Institutional Animal buffer, pH 4.5).

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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 57
Group 2: Metformin (Met): The HFD was fed to rats for 10 performing the histological evaluation was blind to
weeks to produce metabolic syndrome. At the end of 3 week biochemical results and to treatment allocation.
diabetes was induced by a single STZ injection (40 mg/kg 2.7 Statistical Analysis
body wt, i.p. dissolved in 0.01 M citrate buffer, pH 4.5). The The Data were analyzed by One –way analysis of
Metformin (100 mg/kg) was fed orally to rat from 4th weeks variance (ANOVA) and Values were considered at P<0.05.
to 10th weeks daily.
2.6 Evaluation parameters 3. Results
2.6.1 Anthropometric parameter: Body weight (gm), 3.1 Characteristics of the High Fat Diet and low dose of
abdominal circumference (AC), thoracic circumference (TC), STZ induced diabetes with metabolic syndrome:
AC/TC ratio was recorded every 4 weeks and the change in The High Fat diet increased body weight
these parameters were calculated. significantly as compared with the Normal Control at 3rd
2.6.2 Biochemical Parameters: The rat blood samples of all weeks. After 3 weeks of dietary manipulation, rats were
experimental groups were collected from the retro-orbital injected intraperitoneally with STZ (40 mg/kg). The Increased
plexus under light anesthesia at 0, 4, 7 and 10 weeks for Body weight and biochemical parameters (increased Blood
estimation of blood glucose, TC, TG, CPK-MB. In addition, glucose, triglyceride, total cholesterol) resulted in obesity,
after the completion of the experimental duration (10 weeks), dyslipidaemia and type II diabetes, confirmed the diabetes
serum was used for the determination of the following with metabolic syndrome.
parameters like lipid profile, serum insulin, HOMA-IR, 3.2 Anthropometric parameter
HOMA-β, C-peptide, serum DPP-IV, hs-CRP, MDA, The HF-DC and Metformin group showed
pancreatic lipase, SGPT , creatinine by Auto-analyzer or significant (p<0.001) increase in body weight at 4th week as
ELISA kits in the Pathology (NABL accredited) and compared with NC group rats. The increase in body weight in
Pharmacology laboratory. HF-DC and Metformin group rats was not sustained till the
2.6.3 Histopathological studies: At the end of the end of 10th week. Metformin (100 mg/kg) treated group rats at
experiment (10 weeks), the animals were sacrificed. The week 10, showed significant (p<0.01) lower body weight
heart, thoracic aorta, liver, kidney and pancreas were compared to HF-DC group rats. The weight difference
immediately fixed in 10% buffered neutral formalin solution. between NC and HF-DC on Baseline weight and 10th week
The tissues was carefully embedded in molten paraffin with weight was found to be 50.91% in NC, 58% in HF-DC.and
the help of metallic blocks, covered with flexible plastic 32.86 % Metformin. Similarly, the AC and TC of the
moulds and kept under freezing plates to allow the paraffin to Metformin group rats also reduced significantly (p<0.05) only
solidify. Cross sections (5 m thick) of the fixed tissues were at 7th and 10th week as compared to the HF-DC. There was no
cut. These sections were stained with hematoxylin and eosin statistical difference between AC/TC ratio of NC, HF-DC and
and visualized under light microscope to study the Metformin group rats. (Table 1)
microscopic architecture of the tissues. The investigator

Table 1: Anthropometric Parameter

Variable
SN Groups Duration
Body weight AC TC AC/TC
1 NC Baseline 157.63±7.11 14.13±0.49 13.06±00.40 1.081
2 HF-DC 161.14±5.11 14.28±0.39 13.14±0.55 1.08
3 Metformin 159.28±10.48 14.07±0.45 13.00±0.57 1.08
1 NC 4 weeks 188.87±6.22 15.00±0.26 13.93±0.41 1.076
2 HF-DC 235.14±4.59*** 17.72±0.48** 16.71±0.48** 1.06
3 Metformin 230.57±10.59 17.42±0.93 16.35±1.02 1.06
1 NC 7 weeks 214.12±5.33 16.31±0.25 15.25±0.26 1.069
2 HF-DC 226.42±4.68* 17.00±0.40* 16.00±0.41* 1.06
3 Metformin 213.71±11.84$ 15.57 ± 0.73$ 14.47± 0.73$ 1.07
1 NC 10 weeks 237.88±4.99 17.68±0.70 16.62±0.74 1.063
2 HF-DC 219.14±9.92** 16.58±0.45* 15.57±0.47* 1.06
3 Metformin 192.14±10.99$$ 15.35±0.62$ 14.35±0.62$ 1.06
Weight Differences between Baseline (To) and Final ( T 10th week)
Weight Baseline ( T0 wk) Weight Final ( T10th wk) % Difference in weight ( T10th wk- T0 wk/ T0)
NC 157.63 237.88 50.91
HF-DC 161.14 219.14 58
Metformin 159.28 192.14 32.86
NC: Normal Control group (n=8), HF-DC: High fat diabetic control group (n=7) and Met: Metformin group (n=8). Values are
expressed as mean ± SD. *P<0.05, **p<0.01 NC Vs HF-DC,$P<0.05, $$p<0.01HF-DC Vs Met.

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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 58
3.3 Biochemical Parameters: (p<0,001), HOM-IR, and atherogenic index (p<0.01) were
3.3.1 Metabolic Parameters significantly reduced in Metformin group. Serum insulin
The Blood glucose, Triglyceride and Total (p<0.01), and HOMA-β (p<0.01) was significantly increased
Cholesterol levels in the HF-DC group rats were significantly in Metformin group as compared with HF-DC group at the
higher (p<0.001) as compared to NC group rats. In Metformin end of 10th weeks. High density lipoprotein was significantly
group rats these parameters were significantly lower (p<0.05) increased in metformin group rats as compared with
(p<0.001) as compared to HF-DC group rats at 7th and 10th HF-DC. The C-peptide levels in Metformin group increased
week. Glycosylated hemoglobin (p<0,001), Total cholesterol though statistically not significant as compared to NC and
(p<0,001), Triglyceride (p<0.01), Low density lipoprotein HF-DC group rats. (Figure 1,2,3 and Table 2,3)

Figure 1: Time course changes of Blood Glucose level of NC (n=8), HF-DC group (n=7) and Met group (n=8).

450 ***
400 *** ***
Blood glucose (mg/dl)

350
300
250 $$$
$$$
200
150
100
50
0
0 week 4 week 7 week 10 week

Time duration in week

Normal Control HF-Diabetic Control Metformin
*** $$$
Values are expressed as mean±SD. p<0.001 NC Vs HF-DC, P<0.001 HF-DC Vs Met.

Figure 2: Time course changes of HbA1c of NC (n=8), HF-DC group (n=7) and Met group (n=8).

***

$$$

Values are expressed as mean±SD. ***p<0.001 NC Vs HF-DC,$$$P<0.001 HF-DC Vs Met.

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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 59
Figure 3: Time course changes in Total Cholesterol among experimental groups of NC (n=8), HF-DC group (n=7) and
Met group (n=8).
450
Total cholesterol (mg/dl)
400 ***
350
300
250 ***
200
150 ***
$$
100 $$$

50
0
0 week 4 week 7 week 10 week
Time duration in week
Normal Control HF-Diabetic Control Metformin

Values are expressed as mean±SD. ***p<0.001 NC Vs HF-DC,$$P<0.01, $$$P<0.001 HF-DC Vs Met.

Table: 2: Assessment of insulin resistance parameter in various experimental Groups

SN Name of Parameter Insulin C-Peptide HOMA IR HOMB-β
1 NC 6.46±0.65 0.07±0.02 1.57±0.16 66.6±5.86
2 HF-DC 2.93±1.11** 0.05±0.03 2.17±0.63** 5.9±2.2$$
$$
3 Metformin 4.33±1.2 0.073±0.02 1.64±0.5 17.2±4.29$$
NC: Normal Control group (n=8), HF-DC: High fat diabetic control group (n=7) and Met: Metformin group (n=8). Values are expressed as
mean ± SD. **p<0.01 NC Vs HF-DC, $$p<0.01HF-DC Vs Met.
Table 3: Lipid profile in various experimental Groups
SN Variable NC HF-DC Metformin
1 TG (mg/dl) 63.75±11.47 312.85±62.24** 174.14±111.82$$
2 HDL (mg/dl) 32.62±2.56 26.57±5.74* 32.71± 5.05$
***
3 LDL (mg/dl) 12.6±2.41 62.57±12.44 34.82±22.36$$$
**
4 Atherogenic Index 1.36±0.20 11.97±4.76 2.03±0.58$$
NC: Normal Control group (n=8), HF-DC: High fat diabetic control group (n=7) and Met: Metformin group (n=8). Values are expressed as
mean ± SD. *P<0.05, **p<0.01, ***p<0.001 NC Vs HF-DC,$P<0.05, $$p<0.01, $ $$p<0.001HF-DC Vs Met.

3.3.2 Cardiac Variables was a significant (P<0.001) reduction in serum CPK-MB

The CPK-MB levels of different experimental levels in Metformin group rats at 7th and 10th week as
groups did not rise significantly at 4th week However, There compared with HF-DC. (Figure 4)
Figure 4: Time Course Changes in CPK-MB of NC (n=8), HF-DC group (n=7) and Met group (n=8). .
7000
***
6000
CPK-MB (U/L)

5000
***
4000
3000
$ $$$
2000
1000
0
0 week 4 week 7 week 10 week
Time duration in week
Normal Control HF-Diabetic Control Metformin

Values are expressed as mean±SD. ***p<0.001 NC Vs HF-DC,$P<0.05, $$$P<0.001 HF-DC Vs Met.

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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 60
3.3.3 DPP-IV pathway, anti-inflammatory, antioxidant serum DPP-IV level as compared to HF-DC rats. Similarly
Variables inflammatory (hs-CRP) (p<0.05), oxidative marker (MDA)
The serum DPP-IV levels (p<0.001) increased (p<0.01) also significantly reduced in Metformin group as
significantly in HF-DC group rats as compared to NC group compared to HF-DC group rats on 10th week. Hence it shows
rats. Metformin treated rats showed significant reduction in restoring effects in treatment groups. (Table 4)
Table 4: Mechanism; DPP-IV pathway, inflammatory, oxidant Variables in various experimental Groups
SN Name of Parameter NC HF-DC Metformin
1 Serum DPP-IV (microunit/ml) 4.76±0.48 44.53±5.04*** 28.45±3.58$$
2 Hs-CRP (mg/dl) 0.86±0.11 2.2±0.52* 1.11±0.31$
3 MDA (nmol/ml) 1.86±0.08 6.03±0.66*** 3.54±0.08$$
NC: Normal Control group (n=8), HF-DC: High fat diabetic control group (n=7) and Met: Metformin group (n=8). Values are expressed as
mean ± SD. *P<0.05, ***P<0.001 NC Vs HF-DC,$P<0.05, $$ P<0.01HF-DC Vs Met.

3.3.4 Pancreatic, Liver and kidney function markers SGPT (U/L) (p<0.01) and creatinine (mg/dl) (p<0.01) when
The Metformin group rats showed a significant compared to HF-DC group rats at 10th week. (Table 5)
reduced in the level of pancreatic lipase (U/L) (p<0.05),
Table 5: Shows Safety marker in various experimental groups
SN Variable NC HF-DC Metformin
Pancreatic Marker
1 Pancreatic Lipase(U/L) 33.66±4.62 48.26±9.36* 36.83±5.50$
Liver Marker
2 SGPT(U/L) 62.77±11.58 99.85±10.38*** 68.25±6.60$$
Kidney Marker
3 Creatinine (mg/dl) 0.32±0.07 1.27±0.43*** 0.42±0.06$$
NC: Normal Control group (n=8), HF-DC: High fat diabetic control group (n=7) and Met: Metformin group (n=8). Values are expressed as
mean ± SD. *P<0.05, ***p<0.001NC Vs HF-DC,$P<0.05,$$p<0.01 HF-DC Vs Met.
3.4 Histopathological assessment of langerhans, reduced beta cell mass and the atrophy of beta
3.4.1 Histopathological section of myocardium. cells with the loss of few nucleus and cytoplasm,
Photomicrograph of heart of NC group rat heart inflammatory infiltration more was observed (Plate 3B). In
revealed the non-infracted architecture of the myocardium the Metformin treatment group rats Pancreas shows, improve
(Plate 1A).In contrast, HF-Diabetic Control group rat heart beta cell mass less fibrosis, less inflammatory infiltration and
shows fatty infiltration in myocardial cells, hemorrhage, hemorrhage as compared to HF-DC group (Plate 3C). (H&E x
marked edema, confluent areas of myonecrosis separation of 40)
myofibers, congested blood vessels and inflammation as 3.4.4 Histopathological section of Liver
compared to the NC group (Plate 1B). In the Metformin Photomicrograph of Liver sections of NC rats shows,
treatment group rats, occasional focal myofiber loss, normal architecture of central vein, peripheral vein and no
inflammation, necrosis and edema was observed. However congestion of sinosoides (Plate 4A). In contrast, the liver of
the degree of edema, inflammation and necrosis was less as HF-Diabetic Control group rat shows Fatty liver, moderate
compared to the HF-Diabetic Control group (Plate 1C). (H&E fatty degeneration, ballooning of cell, inflammatory
x 40) infiltration more and congestion of blood vessels in central
3.4.2 Histopathological section of Aorta. vein (Plate 4B). In the Metformin treatment group rats Liver
Photomicrograph of aorta sections of Normal control shows, less fatty degeneration, less inflammatory infiltration,
rats shows no histological changes that is, normal alignment congestion of blood vessels, fibrosis, edema and necrosis as
of tunica media, tunica intima, and tunica adventitia as seen compared to HF-DC group (Plate 4C ). (H&E x 40)
(Plate 2A). In case of HF-Diabetic Control group rat aorta 3.4.5 Histopathological section of Kidney
shows, focal and vacuolated cells, inflammation, edema, Photomicrograph of Kidney sections of NC rats
necrosis were seen in aortic layer and atherosclerotic shows normal structure of the kidney. There was absence of
deposition in the vessel wall (Plate 2B). In the Metformin congestion of glomerular blood vessels, tubular necrosis,
treatment group rats aorta shows, well maintain alignment of inflammation and cloudy degeneration (Plate 5A). In contrast
aortic layer with intact intima (Plate 2C). (H&E x 40) histological assessment of the HF-DC group rat demonstrated
3.4.3 Histopathological section of Pancreas congestion of glomerular blood vessels, tubular necrosis,
Photomicrograph of Pancreas sections of NC rats inflammation and cloudy degeneration as compared to NC
shows, an organized pattern and shows normal architecture of group (Plate 5B). In Metformin treated group kidney shows
islets of langerhans and the beta cells (Plate 3A). In contrast, congestion of glomerular blood vessels, less hemorrhage, less
the Pancreas of HF-Diabetic Control group rat shows severe tubular necrosis, inflammation and focal area as compare to
degenerative changes in the pancreatic islets, damaged islets HF-DC group. (Plate 5C) (H&E x 40)
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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 61

Plate 1A Plate 1B Plate 1C

Plate 1:Heart:1A: Photomicrograph of heart of NC group rat heart revealed the non-infracted architecture of the myocardium.
1B: HF-Diabetic Control group rat heart shows fatty infiltration in myocardial cells, hemorrhage, marked edema, confluent
areas of myonecrosis separation of myofibers, congested blood vessels and inflammation. 1C: In the Metformin treatment
group rats, occasional focal myofiber loss and the degree of edema, inflammation and necrosis was less as compared to the HF-
Diabetic Control group.

Plate 2A Plate 2B Plate 2C

AH AH
Plate 2: Aorta:2A: Photomicrograph of aorta sections of NC rats shows no histological changes that is, normal alignment of
tunica media, tunica intima, and tunica adventitia as seen. 2B: In case of HF-Diabetic Control group rat aorta shows, focal and
vacuolated cells, inflammation, edema, necrosis were seen in aortic layer and atherosclerotic deposition in the vessel wall. 2C:
In the Metformin treatment group rats aorta shows, well maintain alignment of aortic layer with intact intima.

Plate 3A Plate 3B Plate 3C

Plate 3: Pancreas:3A: Photomicrograph of Pancreas sections of NC rats shows, an organized pattern and shows normal
architecture of islets of langerhans and the beta cells. 3B: The Pancreas of HF-Diabetic Control group rat shows severe
degenerative changes in the pancreatic islets, damaged islets of langerhans, reduced beta cell mass and the atrophy of beta cells
with the loss of few nucleus and cytoplasm, inflammatory infiltration more was observed. 3C: In the Metformin treatment
group rats Pancreas shows, improve beta cell mass less fibrosis, less inflammatory infiltration and hemorrhage as compared to
HF-DC group.

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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 62

Plate 4A Plate 4B Plate 4C

Plate 4 :Liver: 4A: Photomicrograph of Liver sections of NC rats shows, normal architecture of central vein, peripheral vein
and no congestion of sinosoides.4B: In contrast, the liver of HF-Diabetic Control group rat shows Fatty liver, moderate fatty
degeneration, ballooning of cell, inflammatory infiltration more and congestion of blood vessels in central vein.4C:In the
Metformin treatment group rats Liver shows, less fatty degeneration, less inflammatory infiltration, congestion of blood
vessels, fibrosis, edema and necrosis as compared to HF-DC group.

Plate 5A Plate 5B Plate 5C

Plate 5:Kidney: 5A: Photomicrograph of Kidney sections of NC rats shows normal structure of the kidney. 5B: In contrast
histological assessment of the HF-DC group rat demonstrated congestion of glomerular blood vessels, tubular necrosis,
inflammation and cloudy degeneration as compared to NC group. 5C: In Metformin treated group kidney shows congestion of
glomerular blood vessels, less hemorrhage, less tubular necrosis, inflammation and focal area as compare to HF-DC group.
4. Discussion inflammatory (hs-CRP levels), antioxidant (MDA) and safety
Metformin is an oral antidiabetic drug that has been parameters {pancreas [lipase (U/L)], liver [SGPT (U/L)],
used for decades to reduce plasma glucose, improve insulin renal [creatinine (mg/dl)]} contributing to the beneficial
sensitivity, increase peripheral glucose uptake, and inhibit effects of Metformin in diabetes with metabolic syndrome
hepatic glucose production. Previous studies reported that was studied.
Metformin could improve some of the features of the 4.1 Essential components of Metabolic Syndrome with
metabolic syndrome. Despite these beneficial effects, diabetes:
Metformin’s therapeutic effect in the setting of diabetes with 4.1.1 Diabetes
metabolic syndrome in experimental rats remains unknown. In our study the rats fed with HFD/STZ showed
The major finding of this study is that Metformin significant increase in blood glucose glycosylated
ameliorates diabetes with metabolic syndrome induced hemoglobin, reduced serum insulin levels, which was
deleterious changes in experimental rats. Metformin ameliorated after administration of Metformin treatment at
favorably modulated anti-diabetic (Blood glucose, HbA1c, the end of 10 weeks. The previous study by Latha P. et al.
restoration of pancreatic function), Central obesity (Body (2012) [13] showed similar results by significantly reversed
weight, abdominal circumference (AC), Thoracic the HFD/STZ induced increase in blood glucose levels. The
circumference (TC), AC/TC ratio) and hypolipidemic HOMA-IR was reduced whereas HOMA-β was increased in
(favorable lipid profile, artherogenic index), cardioprotective Metformin treated rats. Study by Dieudonne Kuate et al
(CPK-MB) parameters in the experimental model of diabetes (2015) [14] supported our finding. This observation implied
with metabolic syndrome. Also to understand the that Metformin could exert its hypoglycemic effect through
mechanisms; DPP-IV pathway (serum DPP-IV), anti- improving peripheral Insulin resistance and protecting

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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 63
pancreatic islet β-cells and/or stimulating insulin secretion. all potentiating factors i.e., STZ, ISO and HFD. The
The C-peptide determined by Nora M. El- Sheikh (2012) [15] Myocardial injury induced by High Fat Diet and STZ shown
showed reduced level of C-peptide in diabetic rats similar to by biochemical marker was also confirmed by
present study which is restored by treatment with Metformin. histopathological assessment.
T2DM is characterized by progressive β cell destruction, as a 4.1.5 Mechanism; DPP-IV pathway, inflammatory,
result of chronic IR and the loss of β cell mass and function, oxidant Variables
as demonstrated by previous studies [16]. The primary Many studies involving DPP- IV inhibitors support a
mechanism underlying the reduction in β cell mass in patients principal role for DPP- IV in the inactivation of GLP-1 in
with T2DM is the apoptosis of β cells in all diabetic patients vivo. Due to the recognized benefits of prolonging the
[17]. In patients with T2DM, pancreatic β cells are unable to biological actions of GLP-1, DPP-IV inhibition has been
secrete sufficient quantities of insulin to compensate for the recognized as a possible mechanistic approach to the
reduced insulin sensitivity, which is primarily a result of treatment of type II diabetes [22]. In our study HFD/STZ
insulin secretion dysfunction and a marked reduction in the treated with metformin rats showed reduced serum DPP-IV
number of functional β cells. Consistent with these findings, level in setting of diabetes with metabolic syndrome. Yasuda
the present study observed similar effects, as in the et al in 2002 found that Metformin treatment increased the
Metformin group rats, restoration of β cell mass was observed active levels of GLP-1 in rats, but concluded that this was due
as compared to HF-DC group. to metformin increasing GLP-1 secretion and not a direct
4.1.2 Central obesity inhibitory effect on DPP- IV [23]. However, the Lenhard et al
Various anthropometric parameters such as body in 2004 [24] showed that prolonged dosing of Zucker diabetic
weight, thoracic circumferences (TC), abdominal rats with Metformin reduced serum DPP- IV activity in vivo.
circumference (AC), and their ratios (AC/TC) were evaluated They postulated that prolonged Metformin treatment may
in the Normal control (NC), High Fat Diabetic control (HF- reduce DPP- IV secretion. Similar results found with Lindsay
DC) and Metformin groups. The weight difference between et al in 2005 [25] demonstrated that oral Metformin therapy
NC, HF-DC and Metformin on Baseline weight and 10th week effectively inhibits DPP-IV activity in patients with type II
weight are 50.91% in NC, 58% in HF-DC.and 32.86 % diabetes.
Metformin. Like previous investigators, the weight loss Similar to the present findings Nicolas F. Renna
during Metformin treatment was significant. Similarly, the (2014) [26] also found raised hs-CRP in Fructose fed
anthropometric results may be attributed to the diabetic state Hypertensive rats as compared with normal rats. Similar
that is known to cause weight loss as shown by Dieudonne results were shown by present study. Metformin treated
Kuate et al (2015) [14]. Meanwhile, the present results are significantly reduced in these markers reported by Farnood
consistent with the findings of Gad et al (2010) who reported F1, et al (2014) [27]. Lipid peroxidation, is often used as an
weight reduction by Metformin [18]. index of oxidative tissue damage which causes free radical
4.1.3 Dyslipidemia damage to membrane components of the cell and resulting
The role of dyslipidemia in the development of cell necrosis and inflammation [28]. The end product of
diabetes macrovascular complications is well known. In our oxidative stress revealed that obesity and type II diabetes
study, the HFD/STZ-model of diabetes exhibited enhanced lipid peroxidation. In the present study, MDA was
abnormalities in lipid metabolism as evidenced from the increased significantly in HFD/STZ group. Treatment with
significant elevation of serum TC, TG, LDL-C and reduction Metformin significantly modulated these parameters. Our
of HDL-C levels. These results are in accordance with data are in accordance with the previous report by Kuate et al
previous reports by Siddiqui et al. (2011) [19]. Study by (2015) [14].
Ismail et al (2015) showed treatment of Metformin 4.1.6 Safety variable
significantly reduced the TC, TG, LDL-C level and increased Pancreatic Lipase was assessed to detect pancreatic
HDL-C levels in HFD/STZ rats [20]. Metformin treatment damage. Increased Lipase levels as seen in HFD/STZ rats
also showed favorable effects on atherogenic index. showed presence of pancreatic tissue damage as compared to
4.1.4 Cardiac variable NC. Metformin treated rats restored the architecture of the
The abnormal high levels of CKP-MB is claimed to pancreas. In the present study HFD/STZ treated rats showed
be a specific and extremely sensitive index of myocardial increased levels of SGPT liver enzymes. Numerous studies
necrosis or ischemia. The present study determined the CPK- have reported that diabetes is associated with raised levels of
MB levels to confirm the myocardial injury induced by High SGPT. In a large clinical study reported by Erbey JR et al.,
Fat Diet and STZ in rats. However, treatment of Metformin (2000) [29] patients who were overweight (BMI 25–30
significantly restored increase in serum CPK-MB levels at 7th kg/m2) and obese (BMI > 30 kg/m2) were more likely to
and 10th week. The study by Arshiya Shamim et al (2015) have elevated SGPT levels. There was 10.6% prevalence in
[21] showed increase in the cardiac marker enzymes CPK- obese diabetic patients versus 6.6% prevalence in obese non
MB in diabetic high fat diet rat clearly due to the presence of diabetic patients. Supplementation of Metformin ameliorated

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Rajesh Kumar Suman et al / Metformin ameliorates diabetes with metabolic syndrome induced changes in experimental rats 64
all these changes. Latha et al.(2012) [13] demonstrated [5] Sirtori C. R. and Pasik C. Re-Evaluation of a Biguanide,
similar results by reducing SGPT level in High Fat Diet/ STZ Metformin: Mechanism of Action and Tolerability.
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suggests that diabetic condition is associated with changes in [6] Diabetes Prevention Program Research Group. Reduction
morphology and eventually functional alteration in kidneys. in the incidence of type 2 diabetes with lifestyle
The present results clearly demonstrate increased levels of intervention or metformin. N Engl J Med. 2002;
kidney function marker creatinine in serum of HFD/STZ 346:393–403.
group. In contrast, the HFD/STZ Metformin treated rats [7] Hundal RS, Inzucchi SE. Metformin new understandings,
showed significant reduction in these markers, thus showing new uses. Drugs.2003; 63: 1879–94.
its ability to protect against high fat diet diabetes-induced [8] Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and
kidney damage. Similar were the findings showed by Kuate et energy sensor that maintains energy homeostasis. Nat.
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