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1 Physiology Division, Zoology Department, Faculty of Science, Original Research Article

Beni-Suef University, Beni-Suef, Egypt


2
Received: August 11, 2011
Faculty of Oral and Dental Medicine, Nahda University,
Accepted: March 22, 2012
Beni-Suef, Egypt

ANTIDIABETIC EFFECTS OF HESPERIDIN AND NARINGIN IN


TYPE 2 DIABETIC RATS
Osama M. Ahmed1,2, Ayman M. Mahmoud1*, Adel Abdel-Moneim1, Mohamed B. Ashour1

Key words: hesperidin, naringin, insulin resistance, rats. Both compounds were also found to alleviate
adiponectin, resistin lipid profile and serum adiponectin and resistin levels.
These results showed that hesperidin and naringin
SUMMARY have potential antihyperglycemic and antidyslipidemic
efficacies as well as cardiac function improving action
This study was designed to investigate the effect of in HFD/STZ-induced type 2 diabetic rats.
hesperidin and naringin on serum glucose, blood
glycosylated hemoglobin and serum insulin levels in
INTRODUCTION
high fat fed (HFD)/streptozotocin (STZ)-induced type
2 diabetic rats. In addition, the effect on serum lipid Diabetes mellitus is one of the major health problems
profile, adiponectin and resistin levels, cardiac in both developed and underdeveloped countries.
function parameters and liver and muscle glycogen Diabetes mellitus, a pervasive and multifactorial
contents was assessed. Hesperidin and naringin were metabolic syndrome, is characterized by imperfection
orally and daily administered at a dose level of 50 in insulin secretion and insulin receptor or post
mg/kg b.w. for 30 days to HFD/STZ type 2 diabetic receptor events with derangement in carbohydrate,
rats. Both hesperidin and naringin supplementation protein and lipid metabolism, and results in chronic
potentially ameliorated the elevated levels of glucose, hyperglycemia, a clinical hallmark of diabetes (1).
glycosylated hemoglobin, AST, LDH and CK-MB and Hyperglycemia and hyperlipidemia, as the most
the lowered serum insulin level and hepatic and common features of diabetes mellitus, contribute to
muscle glycogen content of insulin resistant diabetic the development of microvascular and macrovascular
complications of diabetes, which cause the morbidity
Corresponding author: Ayman M. Mahmoud, Physiology Division, Zoology
Department, Faculty of Science, Beni-Suef University, Salah Salim St., and mortality of diabetes (2). In addition, hyper-
P.O. 62511, Beni-Suef, Egypt
E-mail: ayman.mahmoud@science.bsu.edu.eg
glycemia in diabetic patients is associated with
aymano911@yahoo.com alteration in glucose and lipid metabolism and

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O. M. Ahmed, A. M. Mahmoud, A. Abdel-Moneim, M. B. Ashour / ANTIDIABETIC EFFECTS OF HESPERIDIN AND NARINGIN IN
TYPE 2 DIABETIC RATS

modification in liver enzyme levels (3). Diabetes the development of insulin resistance and
mellitus is recognized as a major risk factor for atherosclerosis, and thereby is linked to clinical
cardiovascular diseases (CVD) such as vascular events (23).
atherosclerosis, heart attack, stroke, etc. About 75% of
Nowadays, the agents used as the main means for
deaths among men with diabetes and 57% among
diabetes treatment are synthetic drugs and insulin.
women with diabetes are attributable to CVD (4).
However, these drugs usually come with considerable
Type 2 (non-insulin dependent diabetes mellitus, side effects, such as hypoglycemia, drug resistance,
NIDDM) is a much more prevalent form of diabetes dropsy, and weight gain (24). In contrast, hundreds of
and is responsible for 90% of the disease prevalence traditional folk medicines have demonstrated potential
(5,6). It is caused by insulin resistance coupled by a for the treatment of diabetes with less tolerability and
failure of the beta cell to compensate for it (7). side effects. Thus, there is an increasing need to search
Nowadays, adipose tissue is found to be a site of many for more natural antidiabetic agents from the
polypeptides (adipokines) whose disturbance may lead traditional medicine. Currently, there is much interest
to insulin resistance in type 2 diabetes (8,9). The in the usefulness of citrus fruits and their constituting
disturbance of adipokines may directly or indirectly flavonoids because their intake appears to be
interact with insulin receptors and/or insulin signaling associated with a reduced risk of certain chronic
leading to insulin resistance (10,11). diseases and increased survival, as reported by Chen et
Adiponectin is a peptide hormone predominantly al. (25). However, few publications assessing the
synthesized and secreted from adipose tissue that antidiabetic effects of citrus flavonoids, hesperidin and
modulates a number of metabolic processes, including naringin, were found (26). Thus, the present study was
glucose regulation, fatty acid catabolism, and vascular conducted to evaluate the efficacy of these flavonoids
biology (12,13). In contrast to other adipokines, on the impaired glucose tolerance, insulin resistance
adiponectin is underexpressed in obese patients with and some biochemical parameters of high-fat
insulin resistance or type 2 diabetes mellitus (T2DM) diet/streptozotocin induced-diabetic albino rats and to
(14,15), and in patients with coronary heart disease suggest their probable anti-hyperglycemic and anti-
(15). In humans, circulating levels of adiponectin are hyperlipidemic mechanisms of action.
positively correlated with insulin sensitivity (16). Low
plasma levels of adiponectin (hypoadiponectinemia) MATERIALS AND METHODS
have been observed in several forms of diabetes with
insulin resistance, including T2DM, gestational Chemicals
diabetes, and diabetes associated with lipodystrophy Hesperidin, naringin and streptozotocin were
(16). Resistin belongs to a family of cysteine-rich purchased from Sigma Chemicals Co., St. Louis, MO,
secretory proteins called resistin-like molecules USA, stored at 2-4 C and protected from sunlight. All
(17,18). In rodents, resistin is derived almost other chemicals were of analytical grade and were
exclusively from adipose tissue, and serum resistin is obtained from standard commercial supplies.
elevated in animal models of obesity and insulin
resistance (19,20). Plasma resistin levels were highly
Experimental animals
positively correlated with triglycerides and apoA-
I/apoB ratio, whereas they were inversely correlated White male albino rats (Rattus norvegicus) weighing
with high density lipoprotein (HDL) and apoA-I levels about 19010 g were used as experimental animals in
(21). Moreover, the insulin-resistant effects of resistin the present investigation. The animals were housed in
are thought to account for the activation of glucose 6- standard polypropylene cages and maintained under
phosphatase, which subsequently prevents glycogen controlled room temperature (222 C) and humidity
synthesis and increases the rate of glucose production (555%) with 12-h light and 12-h dark cycle and were
(22). These findings suggest that resistin contributes to fed a standard diet of known composition, and water

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TYPE 2 DIABETIC RATS

ad libitum. The animals used in the present study were group. By the end of the experiment, animals were
maintained in accordance with the principles and sacrificed and blood samples, muscle and liver were
guidelines of the Canadian Council on Animal Care as obtained.
outlined in the Guide for the Care and Use of
Laboratory Animals (27). Biochemical study
On the day before sacrifice, oral glucose tolerance
Development of HFD-fed low dose STZ-treated test (OGTT) was performed in normal, diabetic
type 2 diabetic rats control and diabetic rats treated with hesperidin and
naringin. Blood samples were obtained from lateral
The rats were allocated to two dietary regimens by
tail vein of rats deprived of food overnight (10-12
feeding either normal or high fat diet (HFD) ad
hours). Successive blood samples were then taken at 0,
libitum, for the initial period of 2 weeks (28). The
30, 60, 90 and 120 minutes following the
composition and preparation of HFD have been administration of glucose solution (3 g/kg b.w.)
described elsewhere (29). After 2 weeks of dietary through gastric intubation. Blood samples were left to
manipulation, the group of rats fed HFD were injected coagulate, centrifuged, and clear non-hemolyzed
intraperitoneally (i.p.) with a low dose of STZ (35 serum was obtained for determination of glucose
mg/kg b.w.), while the respective control rats were concentration according to the method of Trinder (30),
given vehicle citrate buffer (pH 4.5) in a dose volume using commercial diagnostic kit (Randox
of 1 mL/kg, i.p. Seven days after STZ injection, rats Laboratories, UK). Serum insulin level was assayed by
were screened for blood glucose levels. Overnight sandwich ELISA using kits purchased from Linco
fasted (10-12 hours) animals were given glucose (3 Research, USA. Blood glycated hemoglobin was
g/kg b.w.) by gastric intubation. After 2 hours of oral determined according to the method of Little et al. (31)
administration, blood samples were taken from lateral using Helena GLYCO-Tek affinity column method
tail vein, left to coagulate and centrifuged; then serum (Helena Laboratories, USA). Liver and muscle
glucose concentration was measured. Rats having glycogen contents were assayed according to the
method of Seifter et al. (32). Serum adiponectin was
serum glucose 200 mg/dL after 2 hours of glucose
assayed by sandwich ELISA using kits purchased from
intake were considered diabetic and selected for
Linco Research (USA) and serum resistin was assayed
further pharmacological studies. The rats were
using ELISA kits purchased from Biovendor (USA).
allowed to continue to feed on their respective diets
until the end of the study. Because abnormalities in insulin action are poorly
detected by a single determination of glucose or
insulin levels (33,34), insulin resistance was evaluated
Experimental design by homeostasis model assessment estimate of insulin
The experimental animals were divided into four resistance (35) as follows:
groups, each group comprising six rats designated as HOMA-IR = fasting insulin level (U/mL) x fasting
follows: group 1 served as control rats; group 2 served blood glucose (mmol/L)/22.5
as diabetic control rats; group 3 served as diabetic rats
Serum cholesterol (36), triglycerides (37), HDL-
administered hesperidin (50 mg/kg b.w.) in aqueous cholesterol (36) and free fatty acids (FFAs) (38) were
suspension orally for 30 days; and group 4 served as estimated. Liver hydroxymethylglutaryl-CoA re-
diabetic rats administered naringin (50 mg/kg b.w.) in ductase (HMG-COA reductase) activity (39) was
aqueous suspension orally for 30 days. The dosage estimated by dividing mevalonate by HMG-COA
was adjusted every week according to any change in (higher ratio means lower activity). Serum LDL-
body weight to maintain similar dose per kg body cholesterol level was calculated from Friedewald (40)
weight of rat over the entire period of study for each formula (LDL-cholesterol = total cholesterol

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TYPE 2 DIABETIC RATS

triglycerides/5 HDL-cholesterol). Serum vLDL- Figure 1. OGTT of normal, diabetic control and
cholesterol concentration was calculated according to diabetic rats treated with hesperidin and naringin. F-
prob.: P<0.001; LSD at 5% level: 7.00; LSD at 5%
Nobert (41) formula (vLDL-cholesterol = tri-
level: 9.44. The means that share the same
glycerides/5). Serum aspartate aminotransferase superscript symbol(s) are not significantly different.
(AST) (42), lactate dehydrogenase (LDH) (43), and
creatine kinase (CK-MB) (44) activities were also
estimated. Cardiovascular indices were calculated
according to Ross formula (45) as follows:
cardiovascular index 1 = total cholesterol/HDL-
cholesterol and cardiovascular index 2 = LDL-
cholesterol/HDL-cholesterol.

Statistical analysis
Data were analyzed using the one-way analysis of
variance (ANOVA) (46) (PC-STAT, University of
Georgia, 1985) followed by LSD test to compare
various groups with each other. Results were
The effect of hesperidin and naringin on the levels of
expressed as mean SE and values of P>0.05 were
fasting serum insulin and blood glycosylated
considered non-significantly different, while those of
hemoglobin (HbA1c%) in HFD/STZ diabetic rats is
P<0.05 and P<0.01 were considered significant and
shown in Table 1. HbA1c% was highly significantly
highly significant, respectively.
elevated (P<0.01; LSD) in diabetic rats as compared
with normal control. Oral administration of hesperidin
RESULTS as well as naringin to diabetic rats significantly
(P<0.01; LSD) improved the altered level; hesperidin
The OGTT of diabetic rats showed a highly seemed to be more effective than naringin. Serum
significant (P<0.01; LSD) elevation in fasting state insulin level, on the other hand, exhibited an opposite
and at 30, 60, 90 and 120 min after oral glucose behavioral pattern; it was significantly (P<0.01; LSD)
loading as compared with normal animals. The decreased in diabetic rats as compared to normal ones
treatment of diabetic animals with hesperidin and and was significantly increased as the result of
naringin induced a potential improvement (P<0.01; treatment with both hesperidin and naringin, which
LSD) of elevated values at all points of OGTT curve had more or less similar effects. Liver and muscle
(Fig. 1).

Table 1. Serum insulin, blood glycosylated hemoglobin (HbA1c%), and liver and muscle glycogen content of
normal, diabetic control and diabetic rats treated with hesperidin and naringin
Parameter Insulin Liver glycogen Muscle glycogen
Group (U/mL) HbA1c% (mg/g tissue) (mg/g tissue)
Normal 26.841.40a 4.710.18d 22.811.86a 4.980.22a
Diabetic control 15.500.76c 8.960.23a 11.950.77c 2.030.22c
Diabetic treated with hesperidin 21.551.13b 5.850.18c 19.331.25b 3.490.18b
Diabetic treated with naringin 20.671.08b 6.260.17b 17.381.14b 3.490.36b
F-prob P<0.001 P<0.001 P<0.001 P<0.001
LSD at 5% 2.32 0.40 2.74 0.53
LSD at 1% 3.17 0.54 3.73 0.72

Data are expressed as mean SE; number of animals in each group is six; means which share the same superscript symbol(s) are not signicantly different.

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TYPE 2 DIABETIC RATS

Table 2. Lipid profile of normal, diabetic control and diabetic rats treated with hesperidin and naringin

Parameter Total cholesterol Triglycerides HDL-cholesterol LDL-cholesterol VLDL-cholesterol Free fatty acids
Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mmol/L)
Normal 67.923.35c 56.842.51c 39.181.37a 17.381.77c 11.030.80c 0.570.07c
Diabetic control 199.905.05a 193.173.28a 26.871.71c 101.195.43a 38.501.79a 1.680.06a
Diabetic treated
93.102.38b 82.682.58b 33.191.65b 42.771.71b 15.971.22b 0.920.10b
with hesperidin
Diabetic treated
98.343.77b 85.712.96b 33.771.53b 46.163.14b 16.241.72b 1.040.12b
with naringin
F-prob P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001
LSD at 5% 7.84 5.93 3.27 7.02 3.00 0.18
LSD at 1% 10.69 8.09 4.46 9.57 4.09 0.25
Data are expressed as mean SE; number of animals in each group is six; means which share the same superscript symbol(s) are not signicantly different.

glycogen contents of HFD/STZ diabetic control rats Figure 2. HOMA-IR index of normal, diabetic control
showed a highly significant decrease (LSD; P<0.01) and diabetic rats treated with hesperidin and
naringin. F-prob.: P<0.001; LSD at 5% level; 0.94; LSD
as compared to normal control. Both treatment agents
at 5% level: 1.28.
showed detectable amelioration of liver and muscle
glycogen contents of diabetic rats (Table 1).
HOMA-IR of normal, diabetic and diabetic treated
with hesperidin and naringin groups is depicted in
Figure 2. Diabetic rats showed a significant (P<0.01;
LSD) elevation of HOMA-IR that was decreased
significantly upon the administration of either
hesperidin or naringin, which had more or less similar
effects.
Data on the effect of hesperidin and naringin on lipid
profile of diabetic rats are presented in Table 2.
Diabetic rats exhibited a highly significant increase
(P<0.01; LSD) in serum cholesterol, triglycerides, Figure 3. Liver hydroxymethylglutaryl-CoA reductase
LDL- and VLDL-cholesterol and FFAs as compared activity (mevalonate/HMG-CoA) of normal, diabetic
control and diabetic rats treated with hesperidin and
with the non-diabetic group. Moreover, HDL-
naringin. F-prob.: P<0.001; LSD at 5% level: 0.34; LSD
cholesterol was affected in an opposite manner, as it at 5% level: 0.47.
was significantly decreased (P<0.01; LSD) in diabetic
rats and significantly increased (P<0.01; LSD) in
response to both treatment agents. The administration
of both hesperidin and naringin led to marked
amelioration of all parameters of the altered lipid
profile. Liver HMG-CoA reductase activity, expressed
as a ratio of HMG-CoA to mevalonate, was
significantly (LSD; P<0.01) increased in diabetic rats
as compared with normal control rats. Administration
of the two tested agents produced a highly significant
(LSD; P<0.01) decrease in the enzyme activity as
compared with diabetic group (Fig. 3).

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TYPE 2 DIABETIC RATS

Table 3. Heart function variables of normal, diabetic control and diabetic rats treated with hesperidin and naringin

Parameter
Group AST (U/L) LDH (U/L) CK-MB (U/L)
Normal 33.621.49c 171.865.71d 111.744.52d
Diabetic control 95.035.24a 276.747.96a 262.245.24a
Diabetic treated with hesperidin 45.071.60b 196.053.84c 180.655.08b
Diabetic treated with naringin 46.792.20b 210.666.43b 168.634.45c
F-prob P<0.001 P<0.001 P<0.001
LSD at 5% 6.35 13.52 10.25
LSD at 1% 8.66 18.44 13.98

Data are expressed as mean SE; number of animals in each group is six; means which share the same superscript symbol(s) are not signicantly different.

Table 3 depicts the effect of hesperidin and naringin Figure 4. Cardiovascular risk indices 1 and 2 of
normal, diabetic control and diabetic rats treated with
administration on some cardiac function biomarkers in
hesperidin and naringin. F-prob.: P<0.001; LSD at 5%
serum of diabetic rats. Serum CK-MB, AST and LDH level: 0.56; LSD at 5% level: 0.75 for cardiovascular
activities were deleteriously increased (LSD; P<0.01) index 1 and F-prob.: P<0.001; LSD at 5% level: 0.24;
LSD at 5% level: 0.58 for cardiovascular index 2.
in diabetic control rats. Moreover, treatment of
diabetic animals with both hesperidin and naringin
induced potential alleviation (LSD; P<0.01) of these
altered parameters; hesperidin seemed to be more
effective than naringin in improving serum AST and
LDH activities, while the latter showed more potent
effect on CK-MB activity. The ratios of total
cholesterol and LDL-cholesterol to HDL-cholesterol
exhibited the same behavioral pattern; they were
highly significantly (LSD; P<0.01) increased in
HFD/STZ diabetic rats as compared to normal control
group. Hesperidin as well as naringin produced
remarkable amelioration of these altered parameters Figure 5. Serum adiponectin of normal, diabetic
(Fig. 4). control and diabetic rats treated with hesperidin and
naringin. F-prob.: P<0.001; LSD at 5% level: 2.11; LSD
Diabetic rats exhibited a highly significant (LSD; at 5% level: 2.88.
P<0.01) decrease in fasting serum adiponectin level as
compared with normal control rats. The administration
of both agents showed marked improvement (LSD;
P<0.01) of serum adiponectin concentration (Fig. 5).
Administration of HFD and STZ produced a highly
significant elevation (P<0.01; LSD) of serum resistin
as compared with normal rats. The treatment of
HFD/STZ diabetic rats with hesperidin and naringin
induced highly significant amelioration (P<0.01;
LSD) of elevated serum resistin (Fig. 6).

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TYPE 2 DIABETIC RATS

Figure 6. Serum resistin of normal, diabetic control Furthermore, Powers (58) states that insulin resistance
and diabetic rats treated with hesperidin and in T2DM causes elevation in blood glucose due to the
naringin. F-prob.: P<0.001; LSD at 5% level: 1.44; LSD
same reasons. From another point of view, the
at 5% level: 2.09.
hyperglycemia observed in our study could be
explained through the glucose-fatty acid cycle (59),
where the high FFAs reduce glucose uptake and
utilization through the increased endogenous glucose
production (60). The present data demonstrated that
the treatment of diabetic rats with either hesperidin or
naringin caused potential amelioration of glucose
tolerance. The decrease in elevated serum glucose
levels is in agreement with the results of Jung et al.
(61), who recorded the anti-hyperglycemic effect of
hesperidin and naringin in C57BL/KsJ-db/db mice.
Moreover, Pari and Suman (62) report on the anti-
hyperglycemic effect of naringin in STZ/nicotinamide
diabetic rats, and Akiyama et al. (63) demonstrated the
DISCUSSION glucose lowering effect of hesperidin in type 1 diabetic
rats.
Type 2 diabetes develops primarily due to insulin
resistance and insulin producing pancreatic -cell The increase observed in the levels of glycosylated
dysfunction, leading to insufficient insulin secretion hemoglobin in diabetic control group rats was due to
(47-49). Feeding rats with HFD can result in insulin- the presence of excessive amounts of blood glucose. In
resistance mainly through Randle or glucose-fatty acid diabetes, the excess of glucose present in blood reacts
cycle (50,51). In our study, HFD/STZ diabetic control with hemoglobin to form glycosylated hemoglobin
rats exhibited significantly elevated fasting blood (64). Estimation of HbA1c has been found to be
glucose and HOMA-IR, accompanied by diminished particularly useful in monitoring the effectiveness of
serum insulin levels. Hence, it is suggested that insulin therapy in diabetes (65). In our study, oral
resistance developed in these animals. Therefore, this administration of hesperidin and naringin significantly
rat model exhibits hyperglycemia and insulin decreased the levels of fasting blood glucose and
resistance that would closely reflect the natural history HbA1c. These results indicated the beneficial effects
and metabolic characteristics of humans, and it is of both hesperidin and naringin in preventing the
further sensitive to pharmacological testing. pathogenesis of diabetic complications caused by
impaired glucose metabolism.
In diabetic animals, the present data indicated a
marked increase in serum glucose levels as compared In comparison with normal control rats, the present
to normal rats. These results run parallel with the study revealed a highly significant decrease in fasting
studies of Schalaan et al. (52) and Ahmed et al. (53). insulin level of HFD/STZ diabetic rats. It can be
Administration of STZ caused rapid destruction of hypothesized that the possible mechanism of
pancreatic -cells in rats, which led to impaired hesperidin and naringin anti-hyperglycemic action
glucose stimulated insulin release and insulin may be through potentiating the pancreatic secretion
resistance, both of which are marked features of of insulin from islet -cells and/or due to enhanced
T2DM (54). Elevation of blood glucose may be transport of blood glucose to peripheral tissue, or by
attributed to the reduced entry of glucose to peripheral other mechanisms such as stimulation of glucose
tissues, muscle and adipose tissue (55), increased uptake by peripheral tissue, inhibition of endogenous
glycogen breakdown (56) and increased glucose production or activation of gluconeogenesis in
gluconeogenesis and hepatic glucose production (57). liver and muscles. By their ability to scavenge free

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TYPE 2 DIABETIC RATS

radicals, hesperidin (66) and naringin (67) prevent et al. (74) demonstrated that the abnormalities in lipid
STZ-induced oxidative stress and protect -cells metabolism generally led to elevation in the levels of
resulting in increased insulin secretion and decrease in serum lipids and lipoproteins, which in turn play an
the elevated blood glucose levels. In this context, Pari important role in the occurrence of premature and
and Suman (62) showed that naringin decreased severe atherosclerosis in patients with diabetes. In the
elevated blood glucose concentration and increased present study, the rise in blood glucose was
insulin release in STZ-induced diabetic rats. Also, accompanied by a marked increase in TC, LDL-C, TG
Akiyama et al. (63) report that in STZ-induced and reduction in HDL-C in HFD/STZ diabetic rats.
diabetic rats, hesperidin decreased blood glucose and These results are in agreement with the findings of Tan
increased serum insulin levels. et al. (75) and Zhang et al. (76), who report increased
Liver glycogen level may be considered as the best serum TG, TC and LDL-C in HFD fed STZ-induced
marker for assessing antihyperglycemic activity of any diabetic rats. On the other hand, HDL-C showed a
drug (68). The increased hepatic glucose output in different behavioral pattern, where it was detectably
diabetes may be derived from glycogenolysis and/or lowered in diabetic rats. Serum HDL-C was found to
gluconeogenesis, as reported by Raju et al. (57). In decrease in HFD/STZ type 2 diabetic rats, as reported
general, increased hepatic glucose production plus by Tan et al. (75) and Schalaan et al. (52), and in
decreased hepatic glycogen synthesis and glycolysis STZ/NA type 2 diabetic rats as shown by Ahmed et al.
are the major symptoms in type 2 diabetes that result (53). Treatment of HFD/STZ diabetic rats with
in hyperglycemia (61). Our results revealed an hesperidin and naringin produced profound
enormous depletion in hepatic and muscle glycogen improvements of the altered serum lipid variables.
contents. These results are in accordance with those of These results are in agreement with the work of
Lavoie and Van de Werve (69) and Ahmed et al. (53), Gorinstein et al. (77), who found that hesperidin and
who found that STZ-induced diabetes reduced hepatic naringin supplementation significantly increased HDL
glycogen content and increased glucose-6- and lowered TC, LDL, total lipids and TG plasma
phosphatase activity in diabetic rats. These results are levels in rats fed a cholesterol-containing diet. The
also in agreement with the work of Grover et al. (68) decrease of LDL levels may occur due to the reduction
and Pari and Suman (62), who demonstrated a of VLDL and the increase of hepatic depuration of
decreased enzymatic activity of hexokinase also in LDL precursors (78). Both hesperidin and naringin
diabetic animals, resulting in depletion of liver significantly ameliorated serum HDL-C in HFD/STZ
glycogen. These changes are obviously due to insulin diabetic rats. That is an advantage, since HDL-C is
deficiency, which in turn results in the activation of responsible for the transportation of cholesterol from
glycogenolytic and gluconeogenic pathways (70). In peripheral tissues to the liver for metabolization. Both
the present study, the elevation of liver glycogen agents thus have the potential to prevent the
content after treatment with hesperidin and naringin development of atherosclerosis and CHD as secondary
was due to amelioration of these altered enzyme complications of severe diabetes mellitus.
activities secondary to the increase of insulin levels in In the HFD/STZ diabetic group, the elevated serum
the blood. FFA level recorded in this study is in agreement with
It is reported that diabetes is associated with that estimated in many previous studies (79,80).
profound alterations in lipid and lipoprotein profile Several mechanisms of how elevated FFA levels
(71). Changes in concentrations of plasma lipids decrease insulin sensitivity have been proposed,
including cholesterol and lipoprotein are including the Randle hypothesis concerning inhibition
complications frequently observed in patients with of insulin-stimulated glucose transport. It also should
diabetes mellitus and certainly contribute to the be noted that FFAs regulate gene expression,
development of coronary heart disease (CHD) in these especially those involved in lipid and carbohydrate
patients (72). In addition, Keenoy et al. (73) and Ravi metabolism (81). Chronically elevated FFAs may also

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TYPE 2 DIABETIC RATS

impair insulin secretory function through toxic effects ideally located for protecting LDL from oxidation
on pancreatic -cells as predicted by the lipotoxicity (61). Moreover, flavonoid consumption was inversely
hypothesis (82). Finally, increased flux of FFAs from associated with mortality from CHD. The relative risks
adipose tissue due to lipolysis of visceral adipose for CHD mortality and first myocardial infarction
depots to the non-adipose tissue (e.g., liver, skeletal were by approximately 50% lower in the highest tertile
muscle) may lead to excessive endogenous glucose of flavonoid intake (89).
production and progression to frank type 2 diabetes
Our study revealed a significant increase in serum
(83). Therefore, decreasing plasma FFA level is
resistin level in HFD/STZ diabetic group in
proposed as a strategy for prevention and treatment of
comparison with that of controls, which ran parallel to
insulin resistance as stated by Na et al. (84). Upon
serum glucose levels, insulin levels and HOMA-IR
treatment of diabetic animals with hesperidin and
index. The findings of this study are in line with that
naringin, there was a decreased level of serum FFA,
of Kushiyama et al. (90), who found that transgenic
which may participate in the insulin sensitizing effects
mice with hepatic resistin overexpression exhibited
of both tested compounds. Also, the ability of
significant hyperglycemia, hyperlipidemia, fatty liver,
scavenging free radicals and antioxidant properties of
and pancreatic islet enlargement when fed a HFD.
both agents may also participate in the hypolipidemic
These effects may be due to resistin-induced
activity of both treatments by inactivating hepatic
HMG-CoA reductase, a key enzyme, in cholesterol impairment of glucose homeostasis and insulin action,
synthesis according to Raz et al. (85), who states that thus modulating one or more steps in the insulin
inhibitors of hepatic HMG-CoA reductase are well signaling pathway and possibly playing a role in the
established drugs for the treatment of hyper- pathogenesis of insulin resistance (91).
cholesterolemia and decrease the incidence of The mechanism whereby resistin decreases insulin
dyslipidemia in diabetic subjects. sensitivity involves several impacts. First, resistin
Diabetic dyslipidemia has long been shown to have a reduces adenosine 5-monophosphate activated protein
strong relation with CHD (65), which is the most kinase activity in skeletal muscle, adipose tissue, and
dangerous and life threatening complication of liver. These alterations decrease tissue insulin
diabetes and the risk of CHD in diabetes increases sensitivity, which results in glucose intolerance,
two- or more folds (86). Increased TG and TC levels elevated FFA levels, and hypertriglyceridemia (22).
and decreased HDL-C represent a displayed lipid Secondly, the resistin-induced reduction in IRS-1 and
profile known as atherogenic profile, which leads to IRS-2 elevates mRNA levels of gluconeogenic
the development of CHD (87). As a favorable effect on enzymes, such as glucose-6-phosphatase and
lipid profile was observed following treatment with phosphoenolpyruvate carboxykinase, thus suggesting
both hesperidin and naringin, this indicated that both a direct resistin induction of insulin resistance in the
agents might help prevent the progression of CVD. In liver (92). Thirdly, resistin decreased glycogen
addition, several atherogenic indices such as TC/HDL- synthase (GS) activity both in the presence and
C and LDL-C/HDL-C have been used to predict CHD absence of insulin; this suggests that resistin directly
risk (88). Reduction of these indices in hesperidin and down-regulates GS activity (93). Furthermore, it has
naringin supplemented diabetic rats strongly been reported that resistin promotes lipid accumu-
supported the notion that dietary supplementation with lation in human macrophages by up-regulating CD36
either hesperidin or naringin may lead to reduction in cell surface expression, which is one of the scavenger
the risk of developing heart diseases. Also, there is receptors in macrophages involved in the uptake of
some evidence to suggest that flavonoids can be modified LDL (94). Based on the current data, the
incorporated into lipoprotein within the liver or resistin lowering effect of hesperidin and naringin may
intestine and subsequently be transported within the directly participate in their anti-hyperglycemic and
lipoprotein particle. Therefore, flavonoids may be anti-hyperlipidemic effects.

Diabetologia Croatica 41-2, 2012 61


O. M. Ahmed, A. M. Mahmoud, A. Abdel-Moneim, M. B. Ashour / ANTIDIABETIC EFFECTS OF HESPERIDIN AND NARINGIN IN
TYPE 2 DIABETIC RATS

In contrast to resistin, HFD/STZ diabetic rats glucose-6-phosphatase (96); and 4) increased fatty
exhibited diminished serum adiponectin level and acid combustion and energy consumption, partly
treatment with either hesperidin or naringin through peroxisome proliferator activated receptor-
significantly alleviated serum adiponectin. Serum activation, leading to decreased TG content in skeletal
levels of adiponectin are found to be in agreement with muscles and liver (11). Moreover, it has been shown
insulin sensitivity and its reduced levels are associated that mice lacking adiponectin expression have reduced
with the etiology of T2DM and obesity (95). Also, insulin sensitivity or are more likely to suffer from
adiponectin has been reported to sensitize body tissues insulin resistance (98). Yet, the insulin sensitizing
toward actions of insulin. The proposed mechanism of effects of the tested flavonoids are mediated partly via
action for adiponectin includes the following: 1) its increasing serum adiponectin level.
insulin sensitizing effect, which in turn regulates
glucose metabolism through stimulation of AMPK Taken together, it can be concluded that the
(96); 2) enhanced oxidation of muscle fat and glucose ameliorative effect of hesperidin and naringin on
transport mediated through AMPK activation and carbohydrate and lipid variables may be attributed to
acetyl-CoA carboxylase inhibition (97); 3) inhibition their insulin releasing capacity, lipid lowering effect,
of hepatic gluconeogenesis through decrease in the and ameliorating the altered adiponectin and resistin
expression of phosphoenolpyruvate carboxylase and levels.

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