Diabetes mellitus is one of the most public metabolic disorders. It is mainly classified into type 1 and type 2. Echinochrome (Ech) is a pigment from sea urchins that has antioxidant, anti-microbial, anti-inflammatory and chelating abilities. The present study aimed to investigate the anti-diabetic mechanisms of Ech pigment in
streptozotocin-induced diabetic rats. Thirty-six male Wistar albino rats were divided into two main groups (18 rats/group). Each group was divided into 3 subgroups (6 rats/subgroup); control, diabetic and Ech subgroups.

Diabetic models were induced by a single dose of streptozotocin (60 mg/kg, i.p) for type 1 diabetes and by a high fat diet for 4 weeks before the injection of streptozotocin (30 mg/kg, i.p) for type 2 diabetes. Diabetic groups were treated orally with Ech (1 mg/kg body weight in 10% DMSO) daily for 4 weeks. Ech groups showed a reduction in the concentrations of glucose, globulins, triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), creatinine, urea, uric acid, malondialdehyde (MDA) and the activities of
arginase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT).

While, it caused general increase in the levels of insulin, total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IB), total protein (TP), albumin, nitric oxide (NO) and the activities of glucose-6-phosphate dehydrogenase (G6PD), hexokinase, glutathione-S-transferase (GST), superoxide dismutase (SOD) and glutathione reduced (GSH). The histopathological investigation showed partial restoration of pancreatic islet cells and clear improvement in the hepatic and kidney architecture.

The results of this study clearly show that Ech has anti-diabetic potential in both types of diabetes. The possible anti-diabetic mechanisms of Ech involving improved glucose metabolism, restoration of β cells, improve insulin secretion, improve insulin signaling and antioxidant activity.

Excerpt

ACKNOWLEDGMENT

First and foremost thanks to God

I would like to express my great appreciation and infinite gratitude to

Prof. Dr. Mohamed Assem Said Marie,

Professor of Environmental

Physiology, Zoology Department, Faculty of Science, Cairo University, for his

supervision, scientific guidance, and continuous help throughout the whole

work.

Special gratitude and sincere thanks to Prof. Dr. Amel Mahmoud

Soliman Professor of Physiology, Zoology Department, Faculty of Science,

Cairo University, for her great assistance, supervision, valuable suggestions and

for his kind help throughout this work and during the preparation of the

manuscript.

I wish to express my deep thanks, grateful acknowledgement and

gratitude to Prof. Dr. Sohair Ramadan Fahmy, Professor of Physiology,

Zoology Department, Faculty of Science, Cairo University for her kind help

during this work. Again, special thanks to staff members and my colleagues of

the Zoology Department, Faculty of Science, Cairo University, for their

encouragement.

Finally, I deeply thank my family for their love, support and

encouragement through the work.

ABSTRACT

Student Name: Ayman Saber Mohamed

Title of the thesis: Metabolic effects of Echinochrome pigment extracted from sea

urchin on diabetic rats

Degree: Ph.D. in Zoology (Molecular and Integrated Physiology)

Diabetes mellitus is one of the most public metabolic disorders. It is mainly

classified into type 1 and type 2. Echinochrome (Ech) is a pigment from sea urchins

that has antioxidant, anti-microbial, anti-inflammatory and chelating abilities. The

present study aimed to investigate the anti-diabetic mechanisms of Ech pigment in

streptozotocin-induced diabetic rats. Thirty-six male Wistar albino rats were divided

into two main groups (18 rats/group). Each group was divided into 3 subgroups (6

rats/subgroup); control, diabetic and Ech subgroups. Diabetic models were induced by a

single dose of streptozotocin (60 mg/kg, i.p) for type 1 diabetes and by a high fat diet

for 4 weeks before the injection of streptozotocin (30 mg/kg, i.p) for type 2 diabetes.

Diabetic groups were treated orally with Ech (1 mg/kg body weight in 10% DMSO)

daily for 4 weeks. Ech groups showed a reduction in the concentrations of glucose,

globulins, triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol

(LDL-C), creatinine, urea, uric acid, malondialdehyde (MDA) and the activities of

arginase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline

phosphatase (ALP) and gamma-glutamyltransferase (GGT). While, it caused general

increase in the levels of insulin, total bilirubin (TB), direct bilirubin (DB), indirect

bilirubin (IB), total protein (TP), albumin, nitric oxide (NO) and the activities of

glucose-6-phosphate dehydrogenase (G6PD), hexokinase, glutathione-S-transferase

(GST),

superoxide dismutase (SOD) and glutathione reduced (GSH). The

histopathological investigation showed partial restoration of pancreatic islet cells and

clear improvement in the hepatic and kidney architecture. The results of this study

clearly show that Ech has anti-diabetic potential in both types of diabetes. The possible

anti-diabetic mechanisms of Ech involving improved glucose metabolism, restoration

of cells, improve insulin secretion, improve insulin signaling and antioxidant activity

Key words: Diabetes-Echinochrome-Oxidative stress-Pancreas-Liver-Kidney-

Histopathology.

List of contents

Page

Title

I. Introduction

Aim of work

II. Materials and methods

II.1. Chemicals and reagents

II.2. Sea urchin Collection

II.3. Echinochrome (Ech) extraction

II.4. Experimental animals

II.5. Ethical Consideration

II.6. Induction of type 1 diabetes mellitus (T1DM)

II.7. Induction of type 2 diabetes mellitus (T2DM)

II.8. Experimental design

II.9. Determination of the physical parameters

II.9.1. Body weight

II.9.2. Urine volume

II.9.3. Hot plate test

II.9.4. Wire suspension

II.10. Animal handling and specimen collection

II.11. Samples preparation

II.11.1. Serum preparation

II.11.2. Liver and kidney homogenate preparation

II.11.3. Histopathological examination

II.12. Biochemical assessment

II.12.1. Diabetic markers

II.12.1.1. Determination of glucose

II.12.1.2. Determination of Insulin

II.12.1.3. Determination of arginase

II.12.1.4. Determination of Hexokinase (HK)

II.12.1.5.

Determination of glucose-6-phosphate

dehydrogenase (G6PDH)

II.12. 2. Serum biomarkers for liver function

II.12.2.1. Determination of serum aminotransferase

enzymes (ASAT, ALAT)

II.12.2.2. Determination of alkaline phosphatase

II.12.2.3. Determination of gamma-glutamyltransferase

(GGT)

II.12.2.4. Determination of total bilirubin, direct &indirect

II.12.2.5. Determination of total protein

II.12.2.6. Determination of serum albumin and globulins

II.12. 3. Determination of lipid profile

II.12.3.1. Determination of serum triglycerides (TG)

II.12.3.2. Determination of serum total cholesterol (TC)

II.12.3.3. Determination

low

density lipoprotein cholesterol

(LDL-C)

II.12.3.4. Determination of High density lipoprotein

cholesterol (HDL-C)

II.12. 4. Determination of kidney Function tests

II.12.4.1. Determination of creatinine and creatinine

clearance

II.12.4.2. Determination of uric acid

II.12.4.3. Determination of urea

II.12. 5. Determination of Oxidative Stress parameters

II.12.5.1.

Determination of lipid peroxide

(Malandialdehyde)

II.12.5.2. Determination

glutathione reduced (GSH)

II.12.5.3. Determination of catalase (CAT)

iii

II.12.5.4. Determination of superoxide dismutase (SOD)

II.12.5.5. Determination

glutathione-S-transferase (GST)

II.12.5.6. Determination of nitric oxide (NO)

II.13. Histological

examination

II.14. Statistical

analysis

III. Results

III.1. Physical parameters

III.1.1. Body weight

III.1.2. Urine volume

III.1.3. Hot plate test

III.1.4. Wire suspension

III.2. Diabetic markers

III.2.1. Serum

glucose

III.2.2. Insulin

III.2.3. Serum

arginase

III.2.4. Liver

hexokinase

III.2.5. Liver glucose-6-phosphate dehydrogenase

(G6PD)

III.3.Serum biomarkers for liver function

III.3.1.

Serum aspartate aminotransferase (AST)

III.3.2.

Serum alanine aminotransferase (ALT)

III.3.3.

Serum alkaline phosphatase (ALP)

III.3.4.

Serum gamma glutamyl transferase (GGT)

III.3.5.

Serum total bilirubin (TB)

III.3.6.

Serum direct bilirubin (DB)

III.3.7.

Serum indirect bilirubin (IB)

III.3.8.

Serum total protein (TP)

III.3.9. Serum

albumin

III.3.10. Serum

globulins

III.3.11.

Albumin/globulins ratio (A/G)

III.4. Lipid profile

III.4.1.

Serum triglycerides (TG)

III.4.2.

Serum total cholesterol (TC)

III.4.3.

Serum low density lipoprotein cholesterol (LDL-

III.4.4. Serum

high

density

lipoprotein cholesterol (HDL-

III.5. Kidney Function tests

III.5.1. Serum

creatinine

III.5.2. Urine

creatinine

III.5.3. Creatinine

clearance

III.5.4.

Serum uric acid

100

III.5.5. Urine

uric

acid

101

III.5.6. Serum

urea

103

III.5.7. Urine

urea

104

III.6. Oxidative stress parameters in liver

104

III.6.1. Liver

malondialdehyde

(MDA)

106

III.6.2.

Liver glutathione reduced (GSH)

107

III.6.3.

Liver catalase (CAT)

109

III.6.4.

Liver superoxide dismutase (SOD)

110

III.6.5. Liver

glutathione-S-transferase

(GST)

112

III.6.6.

Liver nitric oxide (NO)

113

III.7. Oxidative stress parameters in kidney

113

III.7.1.

Kidney malondialdehyde (MDA)

115

III.7.2.

Kidney glutathione reduced (GSH)

116

III.7.3. Kidney

catalase

(CAT)

118

III.7.4.

Kidney superoxide dismutase (SOD)

119

III.7.5.

Kidney glutathione-S-transferase (GST)

121

III.7.6.

Kidney nitric oxide (NO)

122

III.8. Histopathological examination

122

III.8.1.

Histopathological examination of pancreas

124

III.8.2.

Histopathological examination of liver

126

III.8.3.

Histopathological examination of kidney

128

IV. Discussion

167

Conclusion

168

V. Summary

171

VI. References

List of tables

Title

Page

Table 1:

The curative potency of echinochrome (Ech) on the final

body weight (gm) of diabetic rats.

Table 2:

The curative potency of echinochrome (Ech) on urine

volume (ml/24hr) of diabetic rats.

Table 3:

The curative potency of echinochrome (Ech) on hot plate

period (Sec) of diabetic rats.

Table 4:

The curative potency of echinochrome (Ech) on wire

suspension period (Sec) of diabetic rats.

Table 5:

The curative potency of echinochrome (Ech) on glucose

concentration (mg/dl) of diabetic rats.

Table 6:

The curative potency of echinochrome (Ech) on insulin

concentration (µU/ml) of diabetic rats.

Table 7:

The curative potency of echinochrome (Ech) on arginase

activity (U/L) of diabetic rats.

Table 8:

The curative potency of echinochrome (Ech) on hexokinase

activity (U/min/ gm.tissue) of diabetic rats.

Table 9:

The curative potency of echinochrome (Ech) on glucose-6-

phosphate dehydrogenase (G6PD) activity (U/min/

gm.tissue) of diabetic rats.

Table 10:

The curative potency of echinochrome (Ech) on aspartate

aminotransferase (AST) (U/ml) of diabetic rats.

Table 11:

The curative potency of echinochrome (Ech) on alanine

aminotransferase (ALT) activity (U/ml) of diabetic rats.

Table 12:

The curative potency of echinochrome (Ech) on alkaline

phosphatase (ALP) activity (U/L) of diabetic rats.

Table 13:

The curative potency of echinochrome (Ech) on -

glutamyltransferase (GGT) activity (U/L) of diabetic rats.

Table 14:

The curative potency of echinochrome (Ech) on total

bilirubin concentration (mg/dl) of diabetic rats.

Table 15:

The curative potency of echinochrome (Ech) on direct

bilirubin (DB) concentration (mg/dl) of diabetic rats.

Table 16:

The curative potency of echinochrome (Ech) on indirect

bilirubin (IB) concentration (mg/dl) of diabetic rats.

vii

Table 17:

The curative potency of echinochrome (Ech) on total

protein (TP) concentration (g/dl) of diabetic rats.

Table 18:

The curative potency of echinochrome (Ech) on albumin

concentration (g/dl) of diabetic rats.

Table 19:

The curative potency of echinochrome (Ech) on globulins

concentration (g/dl) of diabetic rats.

Table 20:

The curative potency of echinochrome (Ech) on albumin/

globulins (A/G) ratio of diabetic rats.

Table 21:

The curative potency of echinochrome (Ech) on

triglycerides (TG) concentration (mg/dl) of diabetic rats.

Table 22:

The curative potency of echinochrome (Ech) on total

cholesterol (TC) concentration (mg/dl) of diabetic rats.

Table 23:

The curative potency of echinochrome (Ech) on low density

lipoprotein cholesterol (LDL-C) concentration (mg/dl) of

diabetic rats.

Table 24:

The curative potency of echinochrome (Ech) on high

density lipoprotein cholesterol (HDL-C) concentration

(mg/dl) of diabetic rats.

Table 25:

The curative potency of echinochrome (Ech) on serum

creatinine concentration (mg/dl) of diabetic rats.

Table 26:

The curative potency of echinochrome (Ech) on urine

creatinine concentration (mg/dl) of diabetic rats.

Table 27:

The curative potency of echinochrome (Ech) on creatinine

clearance (ml/min) of diabetic rats.

Table 28:

The curative potency of echinochrome (Ech) on serum uric

acid concentration (mg/dl) of diabetic rats.

Table 29:

The curative potency of echinochrome (Ech) on urine uric

acid concentration (mg/dl) of diabetic rats.

100

Table 30:

The curative potency of echinochrome (Ech) on serum urea

concentration (g/dl) of diabetic rats.

102

Table 31:

The curative potency of echinochrome (Ech) on urine urea

concentration (g/dl) of diabetic rats.

103

Table 32:

The curative potency of echinochrome (Ech) on liver

malondialdehyde (MDA) concentration (nmol/g.tissue) of

diabetic rats.

105

viii

Table 33:

The curative potency of echinochrome (Ech) on liver

glutathione reduced (GSH) concentration (mg/g.protein) of

diabetic rats.

106

Table 34:

The curative potency of echinochrome (Ech) on liver

catalase (CAT) activity (U/g. protein) of diabetic rats.

108

Table 35:

The curative potency of echinochrome (Ech) on liver

superoxide dismutase (SOD) activity (U/g. protein) of

diabetic rats.

109

Table 36:

The curative potency of echinochrome (Ech) on liver

glutathione-S-transferase (GST) activity (U/ g. protein) of

diabetic rats.

111

Table 37:

The curative potency of echinochrome (Ech) on liver nitric

oxide (NO) concentration (mol/L) of diabetic rats.

112

Table 38:

The curative potency of echinochrome (Ech) on kidney

malondialdehyde (MDA) concentration (nmol/g. tissue) of

diabetic rats.

114

Table 39:

The curative potency of echinochrome (Ech) on kidney

glutathione reduced (GSH) concentration (mg/ g. tissue) of

diabetic rats.

115

Table 40:

The curative potency of echinochrome (Ech) on kidney

catalase (CAT) activity (U/ g. tissue) of diabetic rats.

117

Table 41:

The curative potency of echinochrome (Ech) on kidney

superoxide dismutase (SOD) activity (U/ g. tissue) of

diabetic rats.

118

Table 42:

The curative potency of echinochrome (Ech) on kidney

glutathione-S-transferase (GST) activity (U/ g. tissue) of

diabetic rats.

120

Table 43:

The curative potency of echinochrome (Ech) on kidney

nitric oxide (NO) concentration (mol/L) of diabetic rats.

121

List of figures

Title

Page

Figure 1:

Formation of advanced glycation endproducts.

Figure 2:

The polyol pathway.

Figure 3:

Relation between NADPH and GSH.

Figure 4:

Synthesis of NO.

Figure 5:

Activation of PKC.

Figure 6:

External shape of Paracentrotus lividus.

Figure 7:

The curative potency of echinochrome (Ech) on the final

body weight (gm) of diabetic rats.

Figure 8:

The curative potency of echinochrome (Ech) on urine

volume (ml/24hr) of diabetic rats.

Figure 9:

The curative potency of echinochrome (Ech) on hot plate

period (Sec) of diabetic rats.

Figure 10:

The curative potency of echinochrome (Ech) on wire

suspension period (Sec) of diabetic rats.

Figure 11:

The curative potency of echinochrome (Ech) on glucose

concentration (mg/dl) of diabetic rats.

Figure 12:

The curative potency of echinochrome (Ech) on insulin

concentration (µU/ml) of diabetic rats.

Figure 13:

The curative potency of echinochrome (Ech) on arginase

activity (U/L) of diabetic rats.

Figure 14:

The curative potency of echinochrome (Ech) on

hexokinase activity (U/min/ gm.tissue) of diabetic rats.

Figure 15:

The curative potency of echinochrome (Ech) on glucose-6-

phosphate dehydrogenase (G6PD) activity (U/min/

gm.tissue) of diabetic rats.

Figure 16:

The curative potency of echinochrome (Ech) on aspartate

aminotransferase (AST) (U/ml) of diabetic rats.

Figue 17:

The curative potency of echinochrome (Ech) on alanine

aminotransferase (ALT) activity (U/ml) of diabetic rats.

Figure 18:

The curative potency of echinochrome (Ech) on alkaline

phosphatase (ALP) activity (U/L) of diabetic rats.

Figure 19:

The curative potency of echinochrome (Ech) on -

glutamyltransferase (GGT) activity (U/L) of diabetic rats.

Figure 20:

The curative potency of echinochrome (Ech) on total

bilirubin concentration (mg/dl) of diabetic rats.

Figure 21:

The curative potency of echinochrome (Ech) on direct

bilirubin (DB) concentration (mg/dl) of diabetic rats.

Figure 22:

The curative potency of echinochrome (Ech) on indirect

bilirubin (IB) concentration (mg/dl) of diabetic rats.

Figure 23:

The curative potency of echinochrome (Ech) on total

protein (TP) concentration (g/dl) of diabetic rats.

Figure 24:

The curative potency of echinochrome (Ech) on albumin

concentration (g/dl) of diabetic rats.

Figure 25:

The curative potency of echinochrome (Ech) on globulins

concentration (g/dl) of diabetic rats.

Figure 26:

The curative potency of echinochrome (Ech) on albumin/

globulins (A/G) ratio of diabetic rats.

Figure 27:

The curative potency of echinochrome (Ech) on

triglycerides (TG) concentration (mg/dl) of diabetic rats.

Figure 28:

The curative potency of echinochrome (Ech) on total

cholesterol (TC) concentration (mg/dl) of diabetic rats.

Figure 29:

The curative potency of echinochrome (Ech) on low

density lipoprotein cholesterol (LDL-C) concentration

(mg/dl) of diabetic rats.

Figure 30:

The curative potency of echinochrome (Ech) on high

density lipoprotein cholesterol (HDL-C) concentration

(mg/dl) of diabetic rats.

Figure 31:

The curative potency of echinochrome (Ech) on serum

creatinine concentration (mg/dl) of diabetic rats.

Figure 32:

The curative potency of echinochrome (Ech) on urine

creatinine concentration (mg/dl) of diabetic rats.

Figure 33:

The curative potency of echinochrome (Ech) on creatinine

clearance (ml/min) of diabetic rats.

Figure 34:

The curative potency of echinochrome (Ech) on serum uric

acid concentration (mg/dl) of diabetic rats.

Figure 35:

The curative potency of echinochrome (Ech) on urine uric

acid concentration (mg/dl) of diabetic rats.

101

Figure 36:

The curative potency of echinochrome (Ech) on serum

urea concentration (g/dl) of diabetic rats.

102

Figure 37:

The curative potency of echinochrome (Ech) on urine urea

concentration (g/dl) of diabetic rats.

104

Figure 38:

The curative potency of echinochrome (Ech) on liver

malondialdehyde (MDA) concentration (nmol/g.tissue) of

diabetic rats.

105

Figure 39:

The curative potency of echinochrome (Ech) on liver

glutathione reduced (GSH) concentration (mg/g.protein) of

diabetic rats.

107

Figure 40:

The curative potency of echinochrome (Ech) on liver

catalase (CAT) activity (U/g. protein) of diabetic rats.

108

Figure 41:

The curative potency of echinochrome (Ech) on liver

superoxide dismutase (SOD) activity (U/g. protein) of

diabetic rats.

110

Figure 42:

The curative potency of echinochrome (Ech) on liver

glutathione-S-transferase (GST) activity (U/ g. protein) of

diabetic rats.

111

Figure 43:

The curative potency of echinochrome (Ech) on liver nitric

oxide (NO) concentration (mol/L) of diabetic rats.

113

Figure 44:

The curative potency of echinochrome (Ech) on kidney

malondialdehyde (MDA) concentration (nmol/g. tissue) of

diabetic rats.

114

Figure 45:

The curative potency of echinochrome (Ech) on kidney

glutathione reduced (GSH) concentration (mg/ g. tissue) of

diabetic rats.

116

Figure 46:

The curative potency of echinochrome (Ech) on kidney

catalase (CAT) activity (U/ g. tissue) of diabetic rats.

117

Figure 47:

The curative potency of echinochrome (Ech) on kidney

superoxide dismutase (SOD) activity (U/ g. tissue) of

diabetic rats.

119

Figure 48:

The curative potency of echinochrome (Ech) on kidney

glutathione-S-transferase (GST) activity (U/ g. tissue) of

diabetic rats.

120

Figure 49:

The curative potency of echinochrome (Ech) on kidney

nitric oxide (NO) concentration (mol/L) of diabetic rats.

122

Figure 50:

Photomicrograph of hematoxylin and eosin stained

pancreas sections.

123

Figure 51:

Photomicrograph of hematoxylin and eosin stained liver 125

xii

sections.

Figure 52:

Photomicrograph of hematoxylin and eosin stained kidney

sections.

127

Figure 53:

The hypoglycemic mechanisms of echinochrome.

166

xiii

List of abbreviation

Abbreviation

Meaning

A/G

Albumin/globulins

AAP

Amino-antipyrine

AAP

Aminophenazone

Ach

Acetylcholine

AchE

Acetylcholine esterase

AGEs

Advanced glycation endproducts

ALP

Alkaline phosphatase

ALT

Alanine aminotransferase

anti-GAD

Anti-glutamic acid decarboxylate

Aldose reductase

AST

Aspartate aminotransferase

BCG

Bromocresol green

CAT

Catalase

Cholesterol esterase

Cholesterol oxidase

CVD

Cardiovascular disorders

DAG

Diacylglycerol

Direct bilirubin

DCHB

Dichloro-2-hydroxybenzenesulfonic acid

DHBS

Dichloro-2-hydroxybenzene sulfonic acid

Diabetes mellitus

DMSO

Dimethyl sulfoxide

DTNB

Dithiobis-2-nitrobenzoic acid

Ech

Echinochrome

G6PDH

Glucose-6-phosphate dehydrogenase

GGT

Gamma-glutamyltransferase

GLUT

Glucose transporter

GOD

Glucose oxidase

GSH

Reduced glutathione

GSH

Glutathione reduced

GST

Glutathione-S-transferase

H&E

Hematoxylin and eosin

HDL-C

High density lipoproteins cholesterol

HFD

High fat diet

Hexokinase

HMOX

Heme oxygenase

IA-2

Insulinoma-associated protein-2

Indirect bilirubin

ICA

Islet cell antibody

xiv

LDL-C

Low density lipoprotein cholesterol

MDA

Malondialdehyde

NAD+

Nicotinamide adenine dinucleotide

NEDA

N-(1-naphthyl)-ethylenediamine

Nitric oxide

NOS

Nitric oxide synthase

PI3K

Phosphatidylinositol-3 kinase

PIs

Phosphatidylinositides

PKC

protein kinase C

PLC

Phospholipase C

PLD

Phospholipase D

POD

Peroxidase

Pancreatic polypeptide

ROS

Reactive oxygen species

SDH

Sorbitol dehydrogenase

SOD

Superoxide dismutase

STZ

Streptozotocin

T1DM

Type 1 diabetes mellitus

T2DM

Type 2 diabetes mellitus

TAGEs

Toxic advanced glycation endproducts

Total bilirubin

TBA

Thiobarbituric acid

Total cholesterol

TCA

Trichloroacetic acid

Triglycerides

Total protein

VLDL

Very low density lipoproteins

I. Introduction

Glucose is an essential metabolic substrate of all mammalian cells. Most

of the energy needed to sustain life is delivered by oxidation of glucose

(Pischetsrieder, 2000). Although glucose is required by all cells, its main

consumer is the brain in the fasting or postabsorptive phase, which accounts for

approximately 50% of the body's glucose use. Another 25% of glucose disposal

occurs in the splanchnic area (liver and gastrointestinal tissue), and the

remaining 25% takes place in insulin-dependent tissues, including muscles and

adipose tissues (DeFronzo, 2004). Approximately 85% of endogenous glucose

production is derived from the liver, with glycogenolysis and gluconeogenesis

contributing equally to the basal rate of hepatic glucose production. The

remaining ~15% of glucose is produced by the kidneys (Mari et al., 1994;

DeFronzo, 2004).

The pancreas is considered as a doubled-entity organ, with both exocrine

and endocrine components, reciprocally interacting with a composed system

whose function is relevant for digestion, absorption, and homeostasis of

nutrients (Piciucchi et al., 2015). Pancreatic islets composed of many types of

cells, including insulin-producing cells, glucagon-releasing cells,

somatostatin-producing cells, pancreatic polypeptide (PP)-containing cells and

ghrelin containing cells (Damasceno et al., 2014). All of these hormones are

involved in the regulation of nutrient metabolism and glucose homeostasis

(Assmann

et al., 2009).

Normally, following glucose ingestion, the increase in plasma glucose

concentration triggers insulin release, which stimulates splanchnic and

peripheral glucose uptake and suppresses endogenous glucose production. In

healthy adults, blood glucose levels are tightly regulated within a range of 70 to

99 mg/dl, and maintained by specific hormones (e.g., insulin, glucagon,

Introduction

incretins) as well as the central and peripheral nervous system, to meet

metabolic requirements (Wardlaw and Hampl, 2007).

Various cells and tissues (within the brain, muscles, gastrointestinal tract,

liver, kidney, and adipose tissue) are also involved in blood glucose regulation

by means of uptake, metabolism, storage, and excretion (DeFronzo, 2004). The

majority of glucose uptake in peripheral tissues occurs in muscles, where

glucose may either be used immediately for energy or stored as glycogen

(Guyton and Hall, 2006). Transport of glucose into muscles is insulin-

dependent, and thus requires insulin for activation of the major enzyme

(glycogen synthase) that regulates production of glycogen (Porte et al., 2003).

While adipose tissue is responsible for a much smaller amount of peripheral

glucose uptake (2%-5%), it plays an important role in the maintenance of total

body glucose homeostasis by regulating the release of free fatty acids (which

increase gluconeogenesis) from stored triglycerides, influencing insulin

sensitivity in the muscles and liver (DeFronzo, 2004). While the liver does not

require insulin to facilitate glucose uptake, it needs insulin to regulate glucose

output (DeFronzo, 2004). So, for example, when insulin concentrations are low,

hepatic glucose output rises (Porte et al., 2003). Additionally, insulin helps the

liver to store most of the absorbed glucose in the form of glycogen (Guyton and

Hall, 2006). The kidneys are increasingly recognized to play an important role

in glucose homeostasis via release of glucose into the circulation

(gluconeogenesis), uptake of glucose from the circulation to meet renal energy

needs, and reabsorption of glucose at the proximal tubule (Wright et al., 2007).

The kidneys also aid in the elimination of excess glucose (when levels exceed

approximately 180 mg/dL, though this threshold may rise during chronic

hyperglycemia by facilitating its excretion in the urine (ADA, 2008).

Introduction

Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in

insulin secretion, insulin action, or both (Kumar and Clark, 2002). Insulin

deficiency, in turn, leads to chronic hyperglycemia with disturbances of

carbohydrate, fat and protein metabolism (Lindberg et al., 2004). DM is

considered as one of the most dangerous metabolic disorders in the world

(Sosale

et al., 2015). It is a complex and potentially debilitating disease that

affects an estimated 8.3% of the adult population or 382 million people

worldwide (IDF, 2013). Egypt will have at least 8.6 million adults with diabetes

and will be the tenth largest population of diabetics in the world (Shaw et al.,

2010). The eleventh most important cause of premature mortality in Egypt is

diabetes mellitus (Saad et al., 2013). It's responsible for 2.4% of all years of life

lost. Also, diabetes is the six most important cause of disability burden in Egypt

(NICHP, 2004).

DM generally classified into type 1 (T1DM) and type 2 (T2DM) diabetes

mellitus. Type 1diabetes (T1DM) is an autoimmune disease, which

characterized by loss of insulin producing cells and reliance on exogenous

insulin for survival (Simmons and Michels, 2015). T1DM is characterized by

mononuclear infiltration of the pancreatic islets, followed by the destruction of

insulin-producing cells (Mathis et al., 2001). The two main forms of clinical

type 1 diabetes are type 1a (about 90% of type 1 cases in Europe) which is

thought to be due to immunological destruction of pancreatic cells, resulting in

insulin deficiency; and type 1b (idiopathic, about 10% of type 1 diabetes), in

which there is no evidence of autoimmunity (Bastaki, 2005). Type 1a is

characterized by the presence of islet cell antibody (ICA), anti-glutamic acid

decarboxylate (anti-GAD) and insulinoma-associated protein-2 (IA-2) that

identify the autoimmune process with cells destruction (Zimmet, et al., 2004).

Autoimmune diseases such as Grave's disease, Hashimoto's thyroiditis and

Addison's disease may be associated with T1a (Atkinson and Maclaren, 1994).

Introduction

There is no known etiological basis for type 1b diabetes mellitus. Some of these

patients have permanent insulinopaenia and are prone to ketoacidosis, but have

no evidence of autoimmunity (McLarty et al., 1990). This form is more

prevalent among individuals of African and Asian origin (Ahrén and Corrigan,

1984).

Mainly, 90-95% of diabetes are diagnosed as T2DM (Leng et al., 2014).

T2DM is increasing in prevalence worldwide (Unwin et al., 2010), and it is

strongly associated with obesity and insulin resistance (Guilherme et al., 2008),

as well as defects in pancreatic cells function and mass (Butler et al., 2003).

T2DM is a multifactorial disease, where the pathophysiology of which involves

not only the pancreas, but also the liver, gastrointestinal tract, adipose tissue and

brain (Cornell, 2015). The cells normally compensate insulin resistance

through more insulin production to keep the glucose near the normal level

(Riguera, 1997). Reduced sensitivity to insulin in liver, muscle, and fatty tissue

lead to impaired insulin secretion and hyperglycemic condition which consider

one of the most characteristic feature of T2DM (Cornell, 2015). Insulin

resistance may be defined as a condition in which normal insulin concentrations

fail to achieve a normal metabolic response (Campbell et al., 1988). Insulin

resistance is usually characterized by a reduction in insulin-stimulated storage of

glucose as glycogen in skeletal muscle and liver (Nielsen, 2008). Insulin

resistance in skeletal muscles is among the earliest detectable defects in humans

with T2DM (Mauvais-Jarvis and Kahn, 2000). Type 2 diabetic patients are

characterized by a decreased fat oxidative capacity and high levels of circulating

free fatty acid (Black et al., 2000). The latter is known to cause insulin

resistance by reducing stimulated glucose uptake most likely via accumulation

of lipid inside the muscle cells (Boden, 1999).

As the disease progresses tissue or vascular damage ensues, leading to

severe diabetic complications such as retinopathy (Bearse et al., 2004),

Introduction

neuropathy (Seki et al., 2004), nephropathy (Shukla et al., 2003),

cardiovascular complications (Saely et al., 2004) and ulceration (Wallace et al.,

2002). Thus, uncontrolled diabetes is implicated in a wide range of

heterogeneous diseases.

Diabetic retinopathy is characterized by a spectrum of lesions within the

retina and is the leading cause of blindness among adults aged 2074 years

(Frank, 2004). These include changes in vascular permeability, capillary

microaneurysms, capillary degeneration, and excessive formation of new blood

vessels (neovascularization). The neural retina is also dysfunctional with death

of some cells, which alters retinal electrophysiology and results in an inability to

discriminate between colors (Forbes and Cooper, 2011).

More than half of all individuals with diabetes eventually develop

neuropathy (Abbott et al., 2011). Diabetic neuropathy affects all peripheral

nerves, including pain fibers, motor neurons and the autonomic nervous system

(Said, 2007). The pathogenesis of diabetic neuropathy is complicated, and the

mechanism of this disease remains poorly understood. It has been suggested that

hyperglycemia is responsible for changes in the nerve tissue (Vinik et al., 1992).

Changes in the blood vessels supplying the peripheral nerves underlie the

mechanisms involved in microvascular damage and hypoxia. Advanced

neuropathy due to nerve fiber deterioration in diabetes is characterized by

altering sensitivities to vibrations and thermal thresholds, which progress to loss

of sensory perception. Pain is also seen in some diabetic individuals without

clinical evidence of neuropathy (1020%), which can seriously impede quality

of life (Obrosova, 2009).

Diabetic nephropathy represents the major cause of end stage renal

failure in Western societies (Gilbertson et al., 2005). Clinically, it is

Introduction

characterized by the development of proteinuria with a subsequent decline in

glomerular filtration rate, which progresses over a long period of time, often

over 1020 years. If left untreated, the resulting uremia is fatal (Forbes and

Cooper, 2011). Importantly, kidney disease is also a major risk factor for the

development of macrovascular complications such as heart attacks and strokes

(Matsushita

et al., 2010). Once nephropathy is established, blood pressure is

often seen to rise, but paradoxically in the short term, there can be

improvements in glycemic control as a result of reduced renal insulin clearance

by the kidney (Amico and Klein, 1981).

Cardiovascular disorders (CVD) accounts for more than half of the

mortality seen in the diabetic population (Haffner et al., 1998), and diabetes

equates to an approximately threefold increased risk of myocardial infarction

compared with the general population (Domanski et al., 2002). In T1DM, it is

not common to see the progression of CVD without impairment in kidney

function (Groop et al., 2009). In T2DM, kidney disease remains a major risk

factor for premature CVD, in addition to dyslipidemia, poor glycemic control,

and persistent elevations in blood pressure (Drury et al., 2011). CVD in

diabetes includes premature atherosclerosis, manifest as myocardial infarction,

stroke as impaired cardiac function well as predominantly diastolic dysfunction

(Forbes and Cooper, 2011).

Oxidative stress has been strongly implicated in the development of

diabetes and diabetic complications (Lupachyk et al., 2013). Metabolic

disorders, assist the increased reactive oxygen species (ROS) production in the

physiological system such as obesity, insulin resistance and diabetes mellitus

(Bhattacharya

et al., 2013). Excess production of ROS can overcome the

antioxidant system of the body, leading to oxidative stress (Ashafaq et al.,

2014). Hyperglycemia condition can induce oxidative stress through many

mechanisms such as glucose autoxidation, polyol pathway, advanced glycation

Introduction

endproducts, (AGEs) formation and protein kinase C (PKC)

(Tangvarasittichai, 2015).

AGEs are modifications of proteins or lipids that become non-

enzymatically glycated and oxidized after contact with aldose sugars (Singh et

al., 2001). Early glycation and oxidation processes, resulting in the formation of

Schiff bases and Amadori products. Further glycation of proteins and lipids

causes molecular rearrangements that lead to the generation of AGEs (Schmidt,

et al., 1994) (Fig.1).

Figure 1: Formation of advanced glycation endproducts.

AGEs may produce ROS, bind to specific cell surface receptors, and

form cross-links (Brownlee et al., 1985). AGEs form in vivo in hyperglycemic

environments and during aging and contribute to the pathophysiology of

vascular disease in diabetes (Schmidt et al., 1995).

AGEs induce link processes

in the structure of long lifespan proteins, such as collagen, modifying blood

vessel structure (Christiane et al., 2014). By binding to their specific receptors,

they activate intracellular signaling pathways which lead to cytokine production,

responsible for the proinflammatory and prosclerotic effects (Inagaki et al.,

Introduction

2003). Toxic advanced glycation endproducts (TAGEs) derived from

glyceraldehydes, are very aggressive compounds and represent the dominant

form of AGEs. The interaction between TAGEs and their receptors in

endothelial and inflammatory cells, leads to intracellular generation of ROS via

the electron transport chain, NADPH oxidase, xanthine oxidase and arachidonic

acid metabolism (Brownlee, 2005).

Aldose reductase (AR) participates in the glucose metabolism by being

the rate-limiting enzyme of the polyol (polyhydric alcohol) pathway. In this

pathway, glucose is reduced to sorbitol by AR, using NADPH as an electron

donor. Sorbitol is subsequently oxidized to fructose by the enzyme sorbitol

dehydrogenase (SDH), using nicotinamide adenine dinucleotide (NAD+) as an

electron acceptor (Forbes and Cooper, 2011) (Fig. 2).

Figure 2: The polyol pathway.

AR acts on many different aldehyde-containing substrates, but sugars

with shorter chains are better substrates than hexoses (Kinoshita, 1990). Due to

its low affinity for glucose, only 3% of all glucose is converted to sorbitol at

normal glucose levels (Ko et al., 1995). Instead, glucose is mainly

phosphorylated into glucose 6-phosphate by hexokinase, which is further

metabolized to pyruvate and lactate via the glycolytic pathway. During

hyperglycemias conditions, however, one third of the glucose is metabolized by

the polyol pathway (Ramana et al., 2003). In addition, the AR activity is

Introduction

enhanced by high glucose concentrations (Kubo et al., 2001). This

hyperglycemia-induced an increase in flux through the polyol pathway has been

proposed to play an important role in the pathogenesis of secondary diabetic

complications in the eyes, nerves and kidneys (Ramana et al., 2003). Although

it is unknown how the increased flux through the polyol pathway is involved in

the occurrence of diabetic disorders several possible mechanisms have been

suggested. One of the first mechanisms proposed is based on the development of

osmotic stress due to sorbitol accumulation (Kinoshita, 1990). Glucose can

easily penetrate the cell membrane while the polyol sorbitol is an osmolyte with

low permeability. Thus, the excessive polyol pathway metabolism results in an

accumulation of sorbitol inside the cells. This causes hypertonicity and osmotic

imbalance that in turn leads to typical diabetic complications, due to swelling

and disruption of the intracellular environment (Kinoshita and Nishimura,

1988)

The most discussed explanation of the deleterious effects of diabetes

caused by a hyperactive polyol pathway is an increased oxidative stress (Lee

and Chung, 1999). When the flux through the polyol pathway is enhanced the

cofactor NADPH is depleted resulting in decreased regeneration of reduced

glutathione (GSH), which also requires NADPH to be formed. GSH is a key

player in this antioxidative system, with a significant function in ROS

scavenging (Noctor et al., 1998) (Fig. 3).

Introduction

Figure 3: Relation between NADPH and GSH.

The depletion of NADPH also affects the production of NO since NO

synthase competes with AR for the same cofactor (Fig.4).

Figure 4: Synthesis of NO.

Decreased levels of NO produce vasoconstriction and slowing of nerve

conduction, which is involved in the pathogenesis of diabetic neuropathy

(Stevens

et al., 1994). It has also been suggested that NOS is inactivated by

enhanced levels of ROS, produced as a result of the hyperglycemia increased

endothelial cell sorbitol concentrations (Gupta et al., 2002).

Introduction

Protein kinase C (PKC) comprises a family of at least 12 isoforms of

serine threonine kinases (Mellor and Parker, 1998). Many in vivo (Shiba et al.,

1993) and in vitro (Inoguchi

et al., 1992) studies have suggested that increased

diacylglycerol (DAG) levels in vascular tissues, is related to PKC activation in

diabetes mellitus. The source of DAG that activates PKC can be derived from

the hydrolysis of phosphatidylinositides (PIs) or from the metabolism of

phosphatidylcholine (PC) by phospholipase C (PLC) or phospholipase D (PLD).

Alternatively, DAG can be synthesized by de novo pathway from glycolytic

intermediates (Park et al., 1999). In metabolic labeling studies, it has been

reported that the incorporation of glucose into the glycerol backbone of the

DAG was increased by hyperglycemia, suggesting that increased DAG contents

were partly derived from de novo pathway (Inoguchi et al., 1994). Recent

studies have identified that the activation of PKC and increased DAG levels are

associated with many vascular abnormalities in retinal, renal, and cardiovascular

tissues (Koya and King, 1998) (Fig. 5).

Figure 5: Activation of PKC.

Introduction

Streptozotocin (STZ) is an antibiotic that was first isolated from the

bacterium Streptomyces achromogens (Vavra et al., 1959). Streptozotocin is

well known for its selective beta cell cytotoxicity, which induces DM in rats

(Mitra

et al., 1996). STZ induced diabetic rat is a suitable model to investigate

the metabolic changes associated with DM as in humans (Ugarte et al., 2012).

The toxic mechanism of STZ is mediated by ROS (Szkudelski, 2001). A single

dose of STZ can induce T1DM in rats (Yin et al., 2006), while T2DM can be

induced by STZ injection after high fat diet (HFD) feeding (Skovso, 2014). The

animal model of high-fat diet, which combined with low dose STZ induced

diabetes manifests many characteristics of human T2DM, such as

hyperglycemia, hyperlipemia and lack of insulin secretion (Yao et al., 2015). In

addition, during HFD/STZ rat model, HFD induces insulin resistance and low

doses of intraperitoneal STZ induce moderate impairment of insulin secretion.

Most of the modern anti-diabetic drugs, including insulin and oral

hypoglycaemic agents only control blood sugar levels as long as they are

regularly administered and are associated with a number of undesirable effects

(Cheng and Caughey, 2007). This generates the need for better, convenient and

less toxic treatment options.

Traditionally, natural products have played an important role in drug

discovery and were the basis of most early medicines (Newman et al., 2000).

The history of the extraction of natural products dates back to the Mesopotamian

and Egyptian times, where production of perfumes, or pharmaceutically - active

oils and waxes were a major business (Bart, 2011). Besides venoms, toxins, and

antibiotic peptides from animals (frogs, spider, snake, etc.), a new focus is

nowadays on the marine world (Bart, 2011). Countless marine plants and

animals contain biochemical secrets that, if unlocked, can provide new insights

and understanding of human diseases and their treatment (Murti and Agrawal,

Introduction

2010).

Marine organisms are a wonderful source of biologically active natural

products (Bhakuni and Rawat, 2005).

Many bioactive compounds have been

extracted from various marine animals (Harvey, 2000). The study of marine

chemical compounds produced by different organisms showed the strategies for

their use for human benefit (Muller et al., 2003). The number of natural

products isolated from marine organisms increases rapidly, and now exceeds

with hundreds of new compounds being discovered every year (Proksch and

Muller, 2006). Chemicals produced by or found in marine organisms have been

shown to have a wide variety of applications as pharmaceuticals for humans and

other animals (Fahmy and Soliman, 2013). These marine chemicals included

antibacterial, analgesic, anti-inflammatory, antimalarial, anticancer, antiparasitic

and antiviral agents (Raval et al., 2013).

A majority of pharmacologically active secondary metabolites have been

isolated from echinoderms (Carballeria et al., 1996). There is much valuable

information for new antibiotic discoveries which give new insights into the

bioactive compounds from the sea urchin (Bragadeeswaran et al., 2013).

Sea urchin (Paracentrotus lividus) is a widespread species in the Atlantic

and the Mediterranean coasts and is subjected to intensive commercial fishing in

several countries (Arafa et al., 2012). Alike many other marine invertebrates,

sea urchins have been considered a source of biologically active compounds

with biomedical applications (Kelly, 2005). However, the potential of echinoids

as a source of biologically active products are largely unexplored

(Bragadeeswaran

et al., 2013).

The sea urchin shells are containing various polyhydroxylated

naphtoquinone pigments, spinochromes (Jeong et al., 2014a) as well as their

analogous compound echinochrome (Ech) which showed bactericidal effect

(Service and Wardlaw, 1984). Ech chemical structure was confirmed by X-ray

Introduction

analysis (Gerasimenko et al., 2006). It is water insoluble compound that

possesses strong antioxidant effects and is the active substance in the drug

Histochrome (Jeong et al., 2014b). Histochrome drug is used in ophthalmic

practice to treat intraocular hemorrhage, diabetic retinopathy, dystrophies,

central retinal vein thrombosis, and post-traumatic hemorrhage in Russia

(Mischenko

et al., 2003). It has been suggested that the phenolic hydroxyl

groups in these molecules could participate in antioxidant activity as was

observed in other well-known antioxidant polyphenols such as tea catechins

(Shankarlal

et al., 2011). The similar compounds are also found in the shells of

sea urchins and thus suggesting that they, as well as echinochrome, would act as

antioxidant substances similar to other polyphenolic antioxidants in edible plants

(Chantaro

et al., 2008). Ech can act through many antioxidant mechanisms,

including the scavenging of active oxygen radicals (Lebedev et al., 2005),

interaction with lipoperoxide radicals (Boguslavskaya et al., 1985), chelation of

metal ions (Lebedev et al., 2008), inhibition of lipid peroxidation (Lebedev et

al., 2008), and regulation of the cell redox potential (Mischenko et al., 2009).

Furthermore, a complex of polyhydroxylated naphthoquinone pigments and

minerals from shells of sea urchins decreases the concentration of blood glucose,

stimulates the synthesis of phospholipids in the liver, and has antioxidant

properties (Kovaleva et al., 2013).

Introduction

Aim of work

The present study carried out to evaluate the antidiabetic efficacy of

echinochrome pigment representing its mechanism in streptozotocin-induced

types I and II diabetic rat models.

Therefore, the present study aimed to include these parts:

The first one aims for the preparing of an echinochrome (Ech) from

shells and spines of sea urchin "Paracentrotus lividus".

The second part includes investigating the effect of Ech on some serum

constituents of both diabetic types including, diabetic markers (glucose, insulin,

arginase, G6PD, hexokinase), liver function markers (ALT, AST, ALP, GGT,

total protein, albumin, direct bilirubin, indirect bilirubin and total bilirubin),

kidney function markers (urea, uric acid, creatinine) and lipid profile

(triglyceride, total cholesterol, LDL-C, HDL-C). Furthermore, evaluate the in

vivo antioxidant effect of Ech through assessing some liver and kidney

parameters include oxidative stress marker such as malondialdehyde (MDA) and

glutathione reduced (GSH) and some antioxidant enzymes such as catalase

(CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST).

The third part aims to evaluate the effect of Ech on pancreas, liver and

kidney architectures using the light microscope.

II.

Materials and methods

II.1. Chemicals and reagents

Streptozotocin, dimethyl sulfoxide (DMSO), insulin and hexokinase kits

were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gamma-

glutamyltransferase (GGT) kit was purchased from Spectrum Diagnostics

company (Obour City, EGY), while the other kits were purchased from the

Biodiagnostic Company (El Motor St, Dokki, EGY).

II.2. Sea urchin Collection

Sea urchins (Paracentrotus lividus) were collected from the

Mediterranean coast of Alexandria (Egypt) (Fig. 6) and transported to the

laboratory packed in ice. The samples were thoroughly washed with sea water to

remove sand and overgrowing organisms at the collection site and transported to

the laboratory. The collected specimens were identified by the standard

literature of taxonomic guide (Clark and Rowe, 1971). The collected specimens

were immediately shade dried.

Figure 6: External shape of Paracentrotus lividus.

Materials and methods

II.3. Echinochrome (Ech) extraction

Pigments in the shells and spines were isolated by the Amarowicz

method with slight modifications (Amarowicz et al., 1994; Kuwahara et al.,

2009). After removal of the internal organs, the shells and spines were washed

with a stream of cold water, air-dried at 4°C for 2 days in the dark and then were

grounded. The powders (5 g) were dissolved by gradually adding 10 ml of 6 M

HCl. The pigments in the solution were extracted 3 times with the same volume

of diethyl ether. The ether layer collected was washed with 5% NaCl until the

acid was almost removed. The ether solution including the pigments was dried

over anhydrous sodium sulfate and the solvent was evaporated under reduced

pressure. The extract including the polyhydroxylated naphthoquinone pigment

was stored at -30°C in the dark.

II.4. Experimental animals

Male albino Wistar rats (Rattus norvegicus) weighing 140 ± 10 gm for

T1DM and 80 ± 10 gm for T2DM were used in this study. The rats were

obtained from the National Research Center (NRC, Dokki, Giza). They were

grouped and housed in polyacrylic cages (six animals per cage) in the well-

ventilated animal house of the Zoology Department, Faculty of Science, Cairo

University. Rats were given food and water ad libitum. Rats were maintained in

a friendly environment of a 12 hr/12 hr light-dark cycle at room temperature (22

C). They were acclimatized to laboratory conditions for 7 days before

commencement of the experiment.

II.5. Ethical Consideration

Experimental protocols and procedures used in this study were approved

by the Cairo University, Faculty of Science, Institutional Animal Care and Use

Committee (IACUC) (Egypt) (CUFS/F/33/14). All the experimental procedures

Materials and methods

were carried out in accordance with international guidelines for the care and use

of laboratory animals.

II.6. Induction of type 1 diabetes mellitus (T1DM)

All rats were starved for 12 hrs before the experiment, but were allowed

free access to water. T1DM was induced by intraperitoneal injection of 60

mg/kg of streptozotocin (STZ) dissolved in 0.1mol/l sodium citrate buffer at pH

4.5. Blood glucose levels were measured 72 hr after injection of STZ using a

blood glucose meter device (ONE TOUCH Ultra 2, USA). Rats were starved,

but had access to drinking water for 6 hrs before blood glucose measurement.

Fasting plasma glucose concentrations 300 mg/100 ml were considered type 1

diabetic in this experiment (Chen et al., 2014).

II.7. Induction of type 2 diabetes mellitus (T2DM)

The rats were fed a high fat diet with energy of 5.3 kcal/g, comprising

60% calories from fat, 35% from protein and 5% from carbohydrate, according

to a modification of the protocols of Reed et al. (2000). After 4 weeks the rats

injected intraperitoneally with a single dose of prepared solution of STZ (30

mg/kg dissolved in 0.1mol/l sodium citrate buffer at pH 4.5). After 72 hours,

fasting plasma glucose concentrations 300 mg/100 ml were considered

diabetic type 2 in this experiment (Ebaid, 2014).

II.8. Experimental design

After one week of acclimatization, 36 rats were assigned into two main

groups (18 rats/group).

x First group: Eighteen rats were randomly assigned into three subgroups

(6 rats/subgroup):

¾ Control subgroup: After a single dose of citrate buffer (0.1mol/l,

i.p), the rats received 1ml (10% DMSO, orally) daily for 4 weeks.

Materials and methods

¾ T1DM subgroup: After a single dose of STZ (60 mg/kg, i.p), the

rats received 1ml (10% DMSO, orally) daily for 4 weeks.

¾ Ech subgroup: After a single dose of STZ (60 mg/kg, i.p), the rats

received 1ml Ech (1mg/kg body weight in 10% DMSO, orally)

(Lennikov

et al., 2014) daily for 4 weeks.

x Second group: Eighteen rats were randomly assigned into three

subgroups (6 rats/subgroup):

¾ Control subgroup: After 4 weeks of normal diets feeding, the rats

injected with a single dose of citrate buffer (0.1mol/l, i.p) then

received 1ml of (10% DMSO, orally) daily for 4 weeks.

¾ T2DM subgroup: After 4 weeks of HFD feeding, the rats injected

with a single dose of STZ (30 mg/kg, i.p) then received 1ml of

(10% DMSO, orally) daily for 4 weeks.

¾ Ech subgroup: After 4 weeks of HFD feeding, the rats injected

with a single dose of STZ (30 mg/kg, i.p) then received 1ml Ech

(1mg/kg in 10% DMSO, orally) daily for 4 weeks.

II.9. Determination of the physical parameters

II.9.1. Body weight

Body weight measured at the beginning and at the end of the

experiments.

II.9.2. Urine volume

Urine collected 24 hrs per-experiment ending using a metabolic cage.

Volume of urine measured, then the urine used for kidney function tests.

Materials and methods

II.9.3. Hot plate test

The hot plate latency was measured using a modification of the original

method of Eddy and Leimbach (1953). Briefly, the modified apparatus consists

of an electric cooking plate (Saiso, Japan) with a 1500 Watts stainless steel

heating element connected to a thermostat (0-400

C); a thermocouple connects

the thermostat to a chrome plated drip pan. The thermocouple together with the

thermostat control the temperature of the hot plate within the desired range once

set. Pain sensitivity was evaluated by the response latency for paw licking on the

hot place. In order to avoid tissue damage, the maximum time the animal could

spend on the hot plate was pegged at 60 seconds. Response latencies were

measured at 15 minute intervals and the average of the results was taken.

II.9.4. Wire suspension

The wire suspension assay measured muscle strength and the prehensile

reflex, an animal's ability to grasp a taut horizontal wire with its forepaws and to

remain suspended. Rats were held gently by the tail and the forepaws were

placed on a suspended wire 2 mm in diameter and 62 cm above a cushioned

surface. The latency to let go was measured using a stopwatch, with shorter

latency to drop indicating reduced strength and/or reflex ability. If a rat did not

let go of the wire within 60 s, it was removed from the wire and a latency of 60 s

was assigned to that measurement. Each rat was used only once in the wire

suspension assay (Troen et al., 2008).

II.10. Animal handling and specimen collection

After the end of all experiments, the rats were fully anesthetized with 3%

sodium pentobarbital, and the chest was opened. A needle was inserted through

the diaphragm and into the heart. Negative pressure was gently applied once the

heart had been punctured, and the needle was repositioned as required until

Materials and methods

blood flowed into the syringe. Blood samples collected in centrifuge tubes.

Liver, pancreas and kidney were removed and immediately blotted using filter

paper to remove traces of blood. Part of the liver and kidney stored at -80

C for

biochemical analysis. Another parts of pancreas, liver and kidney were

suspended in 10% formal saline for fixation preparatory to histopathological

examination.

II.11. Samples preparation

II.11.1. Serum preparation

The blood samples were centrifuged at 3000 rpm for 20 minutes. The

collected serum, stored at -20

C until used for biochemical assays.

II.11.2. Liver and kidney homogenate preparation

Liver and kidney tissues were homogenized (10% w/v) in ice-cold 0.1 M

Tris-HCl buffers (pH7.4). The homogenate was centrifuged at 3000 rpm for 15

min. at 4

C and the resultant supernatant was used for the biochemical analyses.

II.11.3. Histopathological examination

Pancreas, liver and kidney tissues were fixed in 10% neutral-buffered

formalin. The fixed specimens were washed, dehydrated, and embedded in

paraffin wax. The tissues were sectioned at a thickness of 45 µm and stained

with hematoxylin and eosin (HE) according to Bancrof and Stevens (1996),

as routine procedures for histopathological examination.

Materials and methods

II.12. Biochemical assessment

II.12.1. Diabetic markers

II.12.1.1. Determination of glucose

Glucose was determined by colorimetric method using Biodiagnostic

kits, according to the method described by Freund et al. (1986).

Principle:

The enzymatic method uses glucose oxidase (GOD) to catalyze the

oxidation of glucose to hydrogen peroxide and gluconic acid. Hydrogen

peroxide, when combined with 4-aminoantipyrine and a derivative from phenol,

forms a red dye compound. The intensity of the red color produced is directly

proportional to the glucose quantity in the sample. The color intensity is directly

proportional to the protein concentration. It is determined by measuring the

increase in the absorbance at 546 nm.

Reagents:

Standard (ST)

100mg/dl

Phosphate Buffer

100mmol/L

Glucose oxidase

10000 U/L

Peroxidase

2000 U/L

4-AAP

1mmol/L

Phenol

10mmol/L

Procedures:

Blank

Standard

Sample

Reagent (R2)

1.0 ml

Distilled water

10 L

Standard

10 µl

Sample

10 µl

Mix, incubate for 10 minutes at room temperature, measure absorbance

of sample (A

sample

) and standard (A

standard

) against reagent blank within 30

minutes.

Materials and methods

Calculation:

Where 100 is the standard concentration.

II.12.1.2. Determination of Insulin

Insulin was determined by colorimetric method using Sigma-Aldrich

kits, according to the method described by Herbert et al. (1965).

Principle:

The Mouse/Rat insulin ELISA Kit based on the direct sandwich

technique in which two monoclonal antibodies are directed against separate

antigenic determinants on the insulin molecule. During incubation insulin in the

sample reacts with enzyme (HRP)-conjugated anti-insulin antibody and anti-

insulin antibody bound to microplate well. A simple washing step removes the

unbound enzyme labeled antibody. The bound HRP complex is detected by

reaction with TMB substrate.The reaction is stopped by adding acid to give a

colorimetric endpoint that is read using an ELISA reader.

Reagent:

Microwell coated with Insulin MAb

Insulin Standard 1: 1 vial (ready to use)

Insulin Standards 2-6: 5 vials (ready touse)

Insulin Enzyme Conjugate: 1 vial

Assay Diluent: 1 bottle (ready to use)

TMB Substrate: 1 bottle (ready to use)

Stop Solution: 1 bottle (ready to use)

20x Wash concentrate: 1 bottle

Materials and methods

Procedure:

1. Place the desired number of coated strips into the holder.

2. Pipette 25 µL of Insulin standards, control, and sera into appropriate wells.

3. Add 100 µL of working Insulin Enzyme Conjugate to all wells.

4. Thoroughly mix for 10 seconds, it is important to have a complete mixing in

this step.

5. Incubate for 60 minutes at room temperature (1826

C).

6. Remove liquid from all wells. Wash wells three times with 300 µL of 1x wash

buffer. Blot on absorbent paper towels.

7. Add 100 µL of TMB substrate to all wells.

8. Incubate for 15 minutes at room temperature.

9. Add 50 µL of stop solution to all wells. Shake the plate gently to mix the

solution.

10. Read absorbance on ELISA Reader at 450 nm within 15 minutes after

adding the Stopping Solution.

Calculation:

Determine the insulin concentration by using standard curve.

II.12.1.3. Determination of arginase

Arginase was determined using Biodiagnostic kit according to the

method described by Marsch (1965).

Principle:

The method based upon the colorimetric determination of urea by

condensation with diacetyl monoxime in an acid medium in the presence of

ferric chloride and carbazide.

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Details

Title: The Metabolic Effects of Echinochrome Pigment Extracted from Sea Urchin on Diabetic Rats
College: Cairo University (Faculty of Science)
Grade: 4
Author: M.Sc Ayman Mohamed (Author)
Publication Year: 2018
Pages: 243
Catalog Number: V387206
ISBN (eBook): 9783668612952
ISBN (Book): 9783668612969
Language: English
Tags: metabolic effects echinochrome pigment extracted urchin diabetic rats

Quote paper: M.Sc Ayman Mohamed (Author), 2018, The Metabolic Effects of Echinochrome Pigment Extracted from Sea Urchin on Diabetic Rats, Munich, GRIN Verlag, https://www.grin.com/document/387206

The Metabolic Effects of Echinochrome Pigment Extracted from Sea Urchin on Diabetic Rats

Excerpt

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