Pancreas-protective effects of chlorella in STZ-induced diabetic animal model: insights into the mechanism

doi:10.4236/jdm.2011.13006

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Vol.1, No.3, 36-45 (2011)

doi:10.4236/jdm.2011.130 06

Journal of Diabetes Mellitus

Pancreas-protective effects of chlorella in STZ-induced

diabetic animal model: insights into the mechanism

Amr Amin1,5*, Mohamed Lotfy1, Doaa Mahmoud-Ghoneim2,6, Ernest Adeghate3, Mohamed A.

Al-Akhras4, Maryam Al-Saadi1, Salam Al-Rahmoun1, Rasheed Hameed3

1Biology Department, UAE University, Al Ain, United Arab Emirates;

2Physics Department, Faculty of Science, UAE University, Al Ain, United Arab Emirates;

3Department of Anatomy, FMHS; UAE University, Al Ain, United Arab Emirates;

4Department of Physics, Jordan University of Science and Technology, Al Ramtha, Irbid-Jordan;

5Department of Zoology, Faculty of Science, Cairo University, Cairo, Egypt; *Corresponding Author: a.amin@uaeu.ac.ae

6Biophysics Department, Faculty of Science, Cairo University, Cairo, Egypt.

Received 4 May 2011; revised 6 June 2011; accepted 15 June 2011.

ABSTRACT

The aim of this study is to examine the effect of

intragastric administration of chlorella (1 g/kg

body weight) for a period of 30 days to treat

normal and diabetic male Wistar rats. Diabetes

was induced by intraperitoneal injection of

streptozotocin (STZ) (60 mg/kg - 1 body weight).

A significant (p < 0.05) reduction of blood glu-

cose level in diabetic chlorella-treated rats was

observed compared to diabetic untreated. Chlor-

ella increased the number of glut athione-positive

cell in diabetic rats compared to untreated dia-

betics. Chlorella administration increased the

percentage of insulin secreting pancreatic beta

cells both in normal and diabetic treated rats.

Percentage of glucagon producing alpha cells

of the pancreas were reduced both in normal

and diabetic chlorella-treated rats. Chlorella-

induced regenerative ability on pancreas was

mediated by up-regulation of Ki67 and down-

regulation of P53 and by its potent antioxidant

ability. The present results suggest that chlor-

ella may play an important role in improving the

overall condition of diabetic patients and delay

its complication by restoring the function of

pancreatic insulin-secreting cells.

Keywords: Chlorella; Diabetes; Glucose; Insulin;

Beta Cells.

1. INTRODUCTION

Diabetes mellitus is a chronic metabolic disorder af-

fecting approximately 4% of the population worldwide

and it is expected to increase to 5.4% in 2025 [1]. It is

also characterized by hyperglycaemia and associated

with an absolute or relative deficiency in the insulin ac-

tion and/or secretion [2]. The vast majority of cases of

diabetes fall into two broad etiopathogenetic categories.

In type 1 diabetes, patients suffer an absolute deficiency

of insulin secretion. However, in the more prevalent

category type 2 diabetes, the cause is normally a combi-

nation of resistance-to-insulin action and an inadequate

compensatory insulin-secretory response [3,4]. This re-

sults in severe metabolic imbalances and physiologic

changes in many tissues, where oxidative stress plays an

important role in the etiology [5]. The levels of the reac-

tive oxygen species (ROS) as oxidative stress marker in

pancreatic islets of diabetic rats was found to be in-

creased [6].

Oxygen free radical activity can initiate peroxidation

of lipids, which in turn stimulates glycation of protein

and inactivation of enzymes. There is convincing ex-

perimental and clinical evidence that the generation of

ROS is increased in both types of diabetes. Normally,

the level of oxidative stress is modulated by antioxidant

defense systems [7]. Diabetes-linked alterations in anti-

oxidant defense systems have been demonstrated, in-

cluding alteration in the activities of antioxidant en-

zymes such as catalase (CAT) and impaired the reduced

glutathione (GSH) metabolism [8]. Increases in oxida-

tive stress markers in pancreatic islets in experimental

diabetic rats have also been reported [3]. Supplementa-

tion with natural products rich in free radical scavengers

and antioxidants may facilitate the regeneration of

β-cells and protect pancreatic islets against the cytotoxic

effects of streptozotocin [2].

ROS-mediated DNA damage has attracted much at-

tention because of its possible relationship to pathologi-

cal processes such as carcinogenesis and aging [9]. A

role of ROS has also been demonstrated as a cause of

A. Amin et al. / Journal of Diabetes Mellitus 1 (2011) 36-45

3737

type 1 diabetes mellitus induced by chemicals such as

alloxan and STZ in experimental animals [10]. The dia-

betogenic agent STZ enters the pancreatic β-cell via a

glucose transporter-GLUT2 and causes alkylation of

DNA. Furthermore, STZ induces activation of poly-

adenosine diphosphate ribosylation and nitric oxide re-

lease. As a result of STZ action, pancreatic β-cells are

destroyed by necrosis [11]. In adult rats, 60 mg/kg is the

most common dose of STZ used to induce type 1 diabe-

tes [4].

Despite the ongoing introduction of new hypoglyce-

mic drugs, diabetes (and its related complications) re-

mains a major global medical problem and a more suc-

cessful treatment is of great interest, but yet to be dis-

covered. Modern drugs, including insulin and other oral

hypoglycemic agents such as thiazolidinediones, bigua-

nides and sulphonylureas, control blood glucose level as

long as they are regularly administered, but they also

produce many undesirable side effects [1]. Recently,

diabetic healthcare professionals have shown a substan-

tial interest in considering complementary and alterna-

tive approaches to minimize the burden of complications

associated with this disease. Toward that end several

experimental studies have been conducted to evaluate

either raw or purified active ingredients of an array of

natural products. Nowadays, clinical treatment of diabe-

tes targets insulin deficiency, resistance and the declina-

tion of pancreatic β-cell function [4].

Natural antioxidants have gained a great interest of

food scientists, nutrition specialists and even the general

public, as they reduce the risk of chronic diseases and

promote human health [12]. Unicellular green alga chlo-

rella is an eukaryotic microalga. Several lines of evi-

dence have shown the anti-atherogenic effects of chlor-

ella in animal models [13]. Chlorella has also been re-

ported to possess anti-oxidative, anti-inflammatory and

anti-tumor properties both in vitro [14], and in vivo

[15,16]. Administration of chlorella has been shown to

improve glucose challenge both in normal and STZ-

induced diabetic mice [17] and its hypoglycemic effect

was shown both in alloxan-induced and STZ-induced

diabetic animals (STZ) [18].

Despite the chlorella’s protective effects on insulin

secretion, its effect on the integrity of the pancreas of

rats is yet to be carefully investigated in drug-induced

diabetic models. The present study was undertaken to

demonstrate the insulin-conserving potential and preven-

tive nature of chlorella on pancreatic β-cells damage in

STZ-induced diabetic rats, by biochemical, histological

and immunohistochemical techniques.

2. MATERIALS AND METHODS

2.1. Animals and Chlorella

Male Wistar rats weighing approximately 200 grams

(8 weeks old) were used throughout this study. Rats were

obtained from the Faculty of Medicine and Health Sci-

ences, United Arab Emirates University breeding colony.

The study was approved by the UAEU Animal Research

Ethics Committee. All rats were housed at temperature

(25˚C) and humidity controlled rooms and 12 hours light

and dark periods. The animals were fed on a standard rat

chow and tap water ad libitum. Chlorella was purchased

as tablets from Wakunaga of America Co., LTD. Mission

Viejo, CA, USA.

2.2. Induction of Experimental Diabetes

Diabetes was induced in rats by a single intraperito-

neal (ip) injection of STZ (Sigma, Poole, UK) at a dose

of 60 mg/kg body weight [19]. The STZ was freshly

dissolved in citrate buffer (0.5 M, pH 4.5). Five days

after STZ injection, the fifteen hours fasting rats were

tested for diabetes by using a drop of blood from the tail

end of each rat. The blood glucose estimations were

made by One Touch Ultra, Glucometer (LifeScan, John-

son & Johnson, CA, USA) for each individual rat. The

average fasting blood glucose levels of the selected dia-

betic rats were equal to 310 mg/dl.

2.3. Experimental Design

Diabetes Following the diagnosis of diabetes, both age

matched control and STZ-induced diabetic rats were

divided up into four groups each containing ten rats. The

rats were orally administered daily with chlorella (1 g/kg

body weight) for 30 days. The age matched control rats

received the vehicle (water) over the same period of time.

The four groups were as follows: 1) Group 1: normal

untreated control. 2) Group 2: normal chlorella treated

rats. 3) Group 3: diabetic untreated control. 4) Group 4:

diabetic chlorella treated rats.

2.4. Plasma Glucose Measurement

The fasting blood glucose level was measured at

starting time for each individual rat of all groups then

every ten days till the last 30 days. Blood samples for

fasting glucose measurement were drawn from the tail

vein using One Touch Ultra blood glucose meter, Life

Sciences, USA. The mean of the fasting blood glucose

for each of the experimental groups was measured dur-

ing the experiment period.

2.5. Intraperitoneal Glucose Tolerance Test

(IGTT)

At the end of the 30 days of the treatment, all rats in

normal and diabetic groups were subjected to ip glucose

tolerance test, after an overnight fasting (of about 15

A. Amin et al. / Journal of Diabetes Mellitus 1 (2011) 36-45

hours). Each rat was given ip glucose load, 2 g/kg body

weight [20]. The blood glucose measurements were

made at 0 (prior to the glucose load), 30, 60, 120 and

180 min after the glucose load [21].

2.6. Tissue Processing

At the end of the experiment, all the rats from each

group were humanely killed under general anesthesia by

diethyl ether. A mid-line abdominal incision was made

and the pancreas was rapidly removed. Representative

fragments were taken from different parts of the pan-

creas and used for histological studies.

2.7. Histology and Immunohistochemistry

Ten rats from each group (normal and diabetic) were

used for the experiments. The isolated pancreas was

trimmed free of adherent fat and connective tissues, cut

into small pieces (2 mm3), fixed overnight in freshly

prepared 10% neutral phosphate buffered formalin, sec-

tioned at 5 µm thicknesses and stained with hematoxy-

lin-eosin and avidin-biotin-peroxidase complex staining

system. Sections were examined for normal morpho-

logical study and for β-positive cells of insulin secretion

and α-positive cells of glucagon secretion under Olym-

pus BX 41 (Japan) light microscope.

2.8. Immunofluorescence

Isolated pancreatic tissues were retrieved, fixed and

embedded in paraffin. Sections of about 5 µm thickness

were de-paraffinized in xylene, hydrated in descending

concentration of ethanol (3 min each) and washed 3

times in PBS for (5 min each). After washing in PBS, the

tissue was marked around with a Dako pen to prevent

solutions draining away from the tissue section. The

staining procedure was started by incubating the sections

with blocking reagent (bovine serum albumin in phos-

phate buffered saline) for 30 min. Thereafter, the block-

ing reagent was drained off and appropriate dilution of

primary antibodies were applied and incubated at 4˚C

for 24 hours. On the following day, sections were incu-

bated at the room temperature for 1 hour. The slides

were then washed 3 times in PBS (5 min each), incu-

bated with secondary antibodies conjugated with TRITC

(Jackson Laboratory, USA) for 1 hour and washed in

PBS (3 times 5 min each). Sections were finally

mounted in CITI-Floure mounting media and photo-

graphed with Ziess Axiophot Fluorescence Microscope.

2.9. Image Anal ysis

Antigens under investigation were analyzed using

ImageJ software (1.410—by Wayne Rasband, National

Institute of Health, USA). Percentage of antigen posi-

tive-cells in the islets of Langerhans of pancreatic tissue

was determined using an automated image analysis pro-

gram implemented by the authors using Matlab (version

7.00, 1984-2004, The Math Works Inc.). It gives posi-

tive-cells by calculating the percentage ratio of areas

immuno-stained areas with insulin-, glucagon- (DAKO,

USA) while, CAT- and GSH- (Sigma, USA) or with

P53- and Ki67- (Thermo Fisher Scientific, Massachu-

setts, USA) positive cells to the whole islet area on a

slide [22]. The percentages relative to control (which

was set as zero) were calculated. Evaluation was per-

formed in four different islet sites for each of the four

experimental groups (normal untreated, normal chlorella

treated, diabetic untreated and diabetic chlorella treated;

n = 10). The mean and standard error (M ± SEM) value

was then calculated giving the percentage positive cells

for each group.

2.10. Statistical Analysis

All values were expressed as mean ± standard error of

the mean (SEM). Student’s t-test was used to analyze the

significance of differences between mean values and

different groups were assessed using SPSS statistic

analysis software. Values with P < 0.05 were accepted as

significant comparing control and treated samples.

3. RESULTS

3.1. Effect of Chlorella Treatment on Blood

Glucose Levels of Normal and Diabetic

Rats

Figure 1 shows the time course of fasting blood glu-

cose (FBG) level of normal and diabetic rats after treat-

ment with chlorella compared to untreated rats. The

FBG was almost unchanged in normal chlorella treated

rats compared to normal untreated rats. A slight reduc-

tion of FBG levels was detected in diabetic treated rats

compared to untreated diabetic rats.

3.2. Effect of Intraperitoneal Glucose

Tolerance Test on Chlorella Treated

Normal and Diabetic Rats

The results for the IGTT of normal and diabetic rats

were shown in Figure 2. The results showed that normal

rats treated with chlorella had no significant difference

in IGTT from normal untreated rats. However, the dia-

betic chlorella treated rats showed a beneficial effect on

glucose tolerance specially a significant decrease (P <

0.05) in blood glucose levels of diabetic treated rats after

60 min compared to diabetic untreated rats following the

glucose load.

A. Amin et al. / Journal of Diabetes Mellitus 1 (2011) 36-45

3939

Figure 1. Time course effect of chlorella (1 g/Kg body weight)

on blood glucose levels of (a) normal and (b) diabetic rats

compared to untreated control rats. (Data are mean ± SEM, n =

10).

Figure 2. Time course effect of treatment with chlorella (1

g/Kg body weight) on IGTT of (a) normal and (b) diabetic rats

compared to untreated control rats. (*P < 0.05) was a signifi-

cant difference in diabetic treated rats compared to untreated

diabetic rats. (Data are mean ± SEM, n = 10).

3.3. Effect on Antioxidant Enzymes

To assess the potential of chlorella as an antioxidant,

the activities of CAT, and GSH in pancreas were meas-

ured. Figures 3 and 4 depict the levels of activities of

antioxidant enzymes (CAT and GSH), in pancreas of ex-

perimental rats. Diabetic untreated rats showed marked

decrease in activities of antioxidant enzymes, whereas,

diabetic chlorella-treated rats showed a clear elevation of

the activities of antioxidant enzymes significantly com-

pared to diabetic untreated rats.

Panel E of Figures 3-4 and 6-9 show the results of

image analyses for CAT, GSH, insulin, glucagon, Ki67

and P53 immunoreactive cells of pancreatic islets by

immunohistochemical intensity as described in Materials

and Methods.

Figure 3. Representative images of rat pancreatic tissue sec-

tions stained for CAT by immunofluorescence. Micrographs

showing the distribution of CAT positive-cells in the pancreatic

islet of normal and diabetic rats. (a) CAT-positive cells in nor-

mal untreated rat pancreas; (b) CAT-positive cells in normal

chlorella treated rat pancreas; (c) CAT-positive cells in diabetic

untreated rat pancreas; (d) CAT-positive cells in diabetic

treated rat pancreas. Pictures were taken at 400× magnification.

(e) The effect of treatment with chlorella on percentage of

CAT-positive cells in pancreatic islets of normal and diabetic

rats. The data show a significant difference (** P < 0.01) in

diabetic treated rats compared to untreated diabetic rats. (Data

are mean ± SEM, n = 10).

A. Amin et al. / Journal of Diabetes Mellitus 1 (2011) 36-45

3.4. Effect of Chlorella Treatment on

Percentage of Insulin Positive Cells of

Normal and Diabetic Rats

Figure 5 shows a normal histopathological architec-

ture of the pancreatic islets and the ordinary distribution

of pancreatic acini in normal rats. On the other hand, the

diabetic control shows disruption of the acini with small

atrophic islet cells. Treatment of diabetic rats with chlor-

ella leads to expansion and enlargement of islet cells.

Figure 6 shows the effect of chlorella on insulin-

positive cells in normal and diabetic rats. The results

show that treatments of normal and diabetic rats with

chlorella increase the percentage of insulin secreting

positive β-cells in both normal and diabetic treated rats.

However, normal treated rats showed marked signifi-

cant increase (P < 0.01) of insulin secreting positive cells

when compared to normal untreated rats. The results

also showed a significant increase (P < 0.05) of insulin

secreting cells in diabetic treated rats compared to dia-

Figure 4. Representative images of rat pancreatic tissue sec-

tions stained for GSH by immunofluorescence. Micrographs

showing the distribution of GSH positive-cells in the pancre-

atic islet of normal and diabetic rats. (a) GSH-positive cells in

normal untreated rat pancreas; (b) GSH-positive cells in nor-

mal chlorella treated rat pancreas; (c) GSH-positive cells in

diabetic untreated rat pancreas; (d) GSH-positive cells in dia-

betic chlorella treated rat pancreas. Pictures were taken at 400×

magnification. (e) The effect of treatment with chlorella on

percentage of GSH-positive cells in pancreatic islets of normal

and diabetic rats. Results show a significant difference (*P <

0.05) in diabetic treated rats compared to untreated diabetic

rats. (Data are mean ± SEM, n = 10).

betic untreated rats. The histograms also confirmed the

abundance of insulin-positive cells in pancreatic islet of

Langerhan’s in treated rats than in untreated ones in both

normal and diabetic groups.

3.5. Effect of Chlorella Treatment on

Percentage of Glucagon Positive Cells

of Normal and Diabetic Rats

Effects of chlorella on immunoreactive positive α-glu-

cagon cells in normal and diabetic rats were shown in

Figure 7. The results showed that treatments of normal

and diabetic rats with chlorella decrease the percentage

of glucagon secreting positive α-cells in all normal and

diabetic treated rats. A significant (P < 0.001) decrease in

α-cells was detected in diabetic treated rats compared to

diabetic untreated rats. Less prominent α-cells in pan-

creatic islet of Langerhan's in treated normal and dia-

betic rats than in untreated ones was also quantified

(Figure 7(e)).

3.6. Effect of Chlorella Treatment on

Proliferation and Apoptosis in

Pancreatic Islets

In the present study, the expression level of a replication

marker Ki67 was chosen as an established replica-

tion-associated protein with a broad clinical application

(Figure 8). The diabetic-associated significant reduction

(P < 0.01) of Ki67 was alleviated with chlorella treat-

ment.

On the other hand, P53 was chosen as an apoptotic

marker in the pancreatic islets. P53 expression was sig-

Figure 5. Histopathological observation of normal and chlor-

ella-treated rats. (a) Normal (typical architecture of pancreatic

islets); (b) normal treated with chlorella (typical architecture of

pancreatic islets); (c) normal diabetic control (presence of pan-

creatic acini, small atrophic islet cells); (d) diabetic treated

with chlorella (expansion and dilated islet cells). Photos were

taken at 400× magnification.

A. Amin et al. / Journal of Diabetes Mellitus 1 (2011) 36-45

4141

Figure 6. Immunohistochemical evaluation of insulin in pan-

creas. (a) Control rat showing normal structure and insu-

lin-positive β cells; (b) Rats administered with chlorella alone

showing normal structure and insulin-positive β cells; (c)

streptozotocin-diabetic rats showing the decrease in insulin

immunoreactivity and the number of immunoreactive β cells;

(d) Diabetic rats treated with chlorella showing increase of

insulin immunoreactivity and the number of immunoreactive β

cells. Pictures were taken at 400× magnification. (e) The effect

of treatment with chlorella on percentage of insulin-positive

cells in pancreatic islets of normal and diabetic rats. Results

show a significant increase (**P < 0.01 and **P < 0.05) in

normal and diabetic treated rats compared to untreated control

rats. (Data are mean ± SEM, n = 10).

nificantly (P < 0.01) down regulated in the diabetic

chlorella treated group (Figure 9).

4. DISCUSSION

Diabetes mellitus is a severe endocrine disease that

includes a group of disorders of varying etiology and

pathogenesis. The management of diabetes is an esca-

lating global predicament and a cure has yet to be dis-

covered. Drugs such as insulin and other hypoglycemic

agents control blood glucose level only when they are

regularly administered. These treatments, however, have

several disadvantages [23]. Fortunately natural products,

among other alternative medicines, offer a similar degree

of efficacy sans many problematic side effects. STZ is

selectively toxic to the β-cells in the pancreatic islets and

it eventually induces diabetes in adult rats [10].

STZ-induced hyperglycemia has been described as a

good experimental model to study diabetes mellitus. Its

administration to rats showed an increase in the blood

Figure 7. Immunohistochemical evaluation of glucagons in

pancreas. (a) Control rat showing normal structure and gluca-

gon-positive α cells; (b) Rats administered with chlorella alone

showing normal structure and glucagon-positive α cells; (c)

Streptozotocin-diabetic rats showing the increase in glucagon

immunoreactivity and the number of immunoreactive α cells;

(d) Diabetic rats treated with chlorella showing decrease of

glucagon immunoreactivity and the number of immunoreactive

α cells. Pictures were taken at 400× magnification. (e) The

effect of treatment with chlorella on percentage of glucagon

positive cells in pancreatic islets of normal and diabetic rats.

The data show a significant difference (***P < 0.001) in dia-

betic treated rats compared to untreated diabetic rats. (Data are

mean ± SEM, n = 10).

glucose levels and a decrease in the plasma insulin levels

[2].

Hyperglycemia, the most prevalent characteristic of

diabetes mellitus, is in itself very harmful for diabetic

patients. The elevated blood glucose levels are thought

to lead to cell death through oxidative stress induction

that occurs as a common sequel of diabetes-induced

modification of sugar moieties on proteins and lipids

[3,24]. Hyperglycemia increases oxidative stress through

the overproduction of ROS, which results in an imbal-

ance between free radicals and the antioxidant defense

systems of the cells. The administration of chlorella de-

creases blood glucose concentration in diabetic rats

demonstrating the blood glucose-controlling ability of

chlorella and its role as an essential trigger for the liver

to revert to its normal anti-oxidative ability.

Glucose uptake by muscle is increased after chlorella

administration. This may be the explanation for positive

glucose tolerance test in normal mice reported in previ-

A. Amin et al. / Journal of Diabetes Mellitus 1 (2011) 36-45

Figure 8. Immunohistochemical evaluation of Ki67 expression

in pancreas. (a) Control rat showing normal structure and

Ki67-positive β cells; (b) Rats administered with chlorella

alone showing normal structure and Ki67-positive β cells; (c)

streptozotocin-diabetic rats showing the decrease in Ki67 im-

munoreactivity and the number of immunoreactive β cells; (d)

Diabetic rats treated with chlorella showing increase of Ki67

immunoreactivity and the number of immunoreactive β cells.

Pictures were taken at 400× magnification. (e) The effect of

treatment with chlorella on percentage of Ki67-positive cells in

pancreatic islets of normal and diabetic rats. The data show a

significant increase (**P < 0.01) in diabetic treated rats com-

pared to untreated diabetic rats. (Data are mean ± SEM, n =

10).

ous study [17]. Glucose uptakes showed no difference in

soleus muscles between normal and STZ mice, however,

the uptakes were enhanced after chlorella administration.

Therefore, increasing the chlorella-induced glucose

uptake in the soleus muscles may explain its ameliorat-

ing effect on hyperglycemia [18]. At 60 min after intrap-

eritoneal glucose challenge, blood glucose of diabetic

rats treated with chlorella was significantly lower com-

pared to untreated diabetic rats, however, blood glucose

level was not significantly different at other time points.

The inability of the pancreas to significantly lower glu-

cose levels at other time points could be due to the fact

that the newly formed pancreatic beta cells may not be

mature enough to release effective insulin capable of

lowering blood glucose.

Antioxidant protective systems against ROS and the

breakdown products of peroxidized lipids and oxidized

protein and DNA are provided by many enzyme systems

such as CAT and GSH. Oxidative stress in diabetes is

coupled with a decrease in the antioxidant status, which

Figure 9. Immunohistochemical evaluation of P53 expression

in pancreas. (a) Control rat showing normal structure and

P53-positive β cells; (b) Rats administered with chlorella alone

showing normal structure and P53-positive β cells; (c) strepto-

zotocin-diabetic rats showing the increase in P53 immunoreac-

tivity and the number of immunoreactive β cells; (d) Diabetic

rats treated with chlorella showing decrease of P53 immuno-

reactivity and the number of immunoreactive β cells. Pictures

were taken at 400× magnification. (e) The effect of treatment

with chlorella on percentage of P53-positive cells in pancreatic

islets of normal and diabetic rats. The data show a significant

decrease (**P < 0.01) in diabetic treated rats compared to un-

treated diabetic rats. (Data are mean ± SEM, n = 10).

can increase the deleterious effects of free radicals [25].

CAT is one of the two major scavenging enzymes that

remove radicals in vivo. A decrease in its activity can

lead to an excess availability of free radicals like H2O2

which in turn generate resulting in initiation and

propagation of lipid peroxidation [3]. In this study, ac-

tivity of CAT was restored in chlorella treated diabetic

rats compared to diabetic untreated group. In addition,

GSH was significantly increased in diabetic chlorella

treated rats compared to diabetic untreated suggesting

that chlorella has free radical scavenging activity. Chlor-

ella may then enhance the activity of the antioxidant

protective system which may exert a beneficial effect

against pathological alterations caused by reactive oxy-

gen species and thus preserving pancreatic β-cell integ-

rity.

OH 

Changes in blood glucose and in numbers of insu-

lin-secreting cells reflect abnormalities in β-cells func-

tion and structure. STZ impairs glucose oxidation and

decreases insulin biosynthesis and secretion. It also gen-

erates ROS, which contribute to DNA fragmentation and

evoke other deleterious changes in β-cells [1].

A. Amin et al. / Journal of Diabetes Mellitus 1 (2011) 36-45

4343

The present histopathological investigation showed

that diabetic pancreas revealed necrotic and fatty infil-

tration of the islet cells. Chlorella treated diabetic pan-

creas showed near normal structure of pancreatic islet

cells. Also here, chlorella treatment preserved pancreatic

β-cell integrity. The current immunohistochemical ex-

amination showed that pancreatic β-cells are destroyed

by STZ whereas chlorella partially prevented degenera-

tion of β-cells. Chlorella treatment increased the area of

insulin immunoreactive β-cells significantly. In the

STZ-diabetic rats, a significant decrease in insulin im-

munoreactivity was observed. However, after the treat-

ment of chlorella, the insulin immunoreactivity was im-

proved and an increase in the number of immunoreactive

β-cells was observed in comparison to the diabetic group.

According to the immunohistochemical results obtained,

chlorella may have the ability to enhance insulin sensi-

tivity and regenerate the β-cells of diabetic rats.

Anti-hyperglycemic effect of natural product extracts is

generally dependent upon the degree of β-cell destruc-

tion [26]. The present findings suggest that chlorella

may regenerate β-cells and has protective effect on

β-cells from glucose toxicity since the morphological

changes seen in β-cells of diabetic rats are compatible

with those observed when islets are exposed to high

concentrations of glucose. The anti-hyperglycemic effect

of chlorella may be mediated through insulin secretion

from the remnant β-cells and increase insulin sensitivity

[27]. Aucubin, another natural product isolated from

Plantago asiatica, was shown to stimulate glucose in-

take into cells and thus lowering the blood glucose level

[3]. A number of other natural products have also been

observed to exert hypoglycemic activity through insulin

release stimulatory effects [1]. Similarly, administration

of chlorella to diabetic rats markedly reduced STZ-in-

duced number of glucagon-positive cells in the pancreas.

In the present study, the expression level of Ki67 was

chosen as an established replication-associated protein

with a broad clinical application, e.g. in the histological

grading of malignant tumours. Previous studies reported

tight correlations between Ki67 and many other replica-

tion markers in a variety of tumours and suggested that

Ki67 is suitable for the quantitative determination of

β-cell replication in pancreatic tissue sections [28].

Even though the overall expression Ki67 was signifi-

cantly up regulated following chlorella treatment in dia-

betic rats, the expression of Ki67 protein was restricted

within individual cells of the pancreatic islets. This sug-

gests that the expression of other proliferation markers

may indeed be associated with the process of β-cell rep-

lication. In this context, MCM7 protein has been de-

scribed to peak at the G1/S-transition point in various

cell types [29], while PCNA expression reaches its

maximum during S-phase [30]. Ki67 is expressed at very

low levels in late G1 and early S [31] but increases dur-

ing cell cycle progression, reaching its maximum in late

S and G2 [32]. By these means, determination of the

expression of more than one replication marker may not

only allow for the discrimination between replicating

cells and quiescent cells, but may also yield additional

information about its current stage of cell cycle progres-

sion [28].

In this study, we also demonstrate that chlorella at-

tenuates apoptosis and increases cell survival in pancre-

atic β-cells. This effect was mediated by down regulating

p53. Pancreatic β-cell failure plays a key role in the

pathogenesis of type 2 diabetes. Although the exact

mechanism underlying β-cell destruction is not known, it

has been suggested that both hyperglycemia and hyper-

lipidemia contribute to β-cell destruction. Both glu-

cotoxicity and lipotoxicity are important in the patho-

genesis of type 2 diabetes because they lead to interfer-

ence with insulin signal transduction and thus insulin

resistance on the one hand and β-cell destruction on the

other [33]. The functional consequences of glucotoxicity

and glucolipotoxicity for β-cells include induction of cell

death by apoptosis [34].

The present study suggests that the antihyperglycemic

and antioxidant effects of chlorella preserve endocrine

pancreas and protect pancreas from STZ-induced toxic-

ity. Increased insulin secreting cells after treatment with

chlorella positively altered the deranged carbohydrate

metabolism in the STZ-induced diabetic rats. The pro-

tective effects of chlorella in experimental diabetes are

mediated by decreasing oxidative stress, enhancing pro-

liferation and cell survival and resulting in the preserva-

tion of pancreatic β-cell integrity.

5. ACKNOWLEDGEMENTS

This study was partially supported by the Dean’s Award for Prof. A.

Amin, Faculty of Science, UAEU. Authors are grateful to Sayel Daoud

(Twaam Hospital) for his technical assistance.

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