A new approach in Type 2 diabetes mellitus treatment: Evaluation of the beneficial effect of L-cysteine in the treatment of type 2 diabetes mellitus
©2015
Textbook
246 Pages
Summary
Type 2 diabetes mellitus (T2DM) is a chronic, progressive metabolic disease characterized by chronic hyperglycemia. Although its main physiological abnormalities are insulin resistance and impaired insulin secretion, the specific underlying determinants of these metabolic defects remain uncertain. <br>There are complex interactions between genetic, epigenetic, environmental and behavioral factors that contribute to the development of diabetes. Non-pharmacological and pharmacological interventions have been used for diabetic management. Over the past few years, research has started to focus on the use of novel adjuvant drugs as antioxidants and anti-inflammatory drugs for better management, as it was revealed that both oxidative stress and inflammation play a critical role in the disease pathogenesis.<br>Thus, the development of antidiabetic drugs that can reverse insulin resistance is a potential therapeutic target. Although antidiabetic drugs may be effective in improving glycemic control, they do not appear to be effective in entirely preventing the progression of pancreatic ß-cells damage mediated by chronic hyperglycemia-induced decline in intracellular antioxidants. Therefore, antioxidant and anti-inflammatory therapy should be considered as an adjunct to the commonly used oral antidiabetics
Excerpt
Table Of Contents
iv
LIST OF FIGURES
Figure
Page
(1)
Model for the effects of adipocytes on pancreatic -cell
function/mass and insulin sensitivity in the pathogenesis of
type 2 diabetes
10
(2)
Mitochondrial overproduction of superoxide activates the
major pathways of hyperglycemic damage by inhibiting
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
16
(3)
Development of type 2 diabetes
19
(4)
The cellular origins of reactive oxygen species, their targets,
and antioxidant systems.
23
(5)
Schematic of the effects of chronic oxidative stress on the
insulin signaling pathway
25
(6)
Structure of GSH ( -glutamylcysteinyl glycine), where the N-
terminal glutamate and cysteine are linked by the -carboxyl
group of glutamate
29
(7)
Chemical structure of L-cysteine
32
(8)
The transsulfuration pathway in animals.
33
(9)
Sources and actions of cysteine and glutathione (GSH)
36
(10)
The role of serine kinase activation in oxidative stress-
induced insulin resistance and the protective effect of some
antioxidants by preserving the intracellular redox balance
37
(11)
Chemical structure of biguanides
41
(12)
Structure of human proinsulin and some commercially
available insulin analogs.
57
(13)
Model of control of insulin release from the pancreatic -cell
by glucose and by sulfonylurea drugs
59
(14)
Schematic diagram of the insulin receptor heterodimer in the
activated state.
60
(15)
Standard curve of insulin
73
(16)
Standard curve of Monocyte chemoattractant protein-1
(MCP-1)
82
(17)
Standard curve of nitric oxide
85
(18)
Standard curve of MDA
88
(19)
Standard curve of reduced glutathione
91
(20)
Standard curve of Protein
93
(21)
Effect of STZ-induced type 2 diabetes on fasting serum
glucose in male albino rats
104
v
Figure
Page
(22)
Effect of STZ-induced type 2 diabetes on fasting serum
insulin in male albino rats
104
(23)
Effect of STZ-induced type 2 diabetes on HOMA-IR in male
albino rats
104
(24-a)
Effect of STZ-induced type 2 diabetes on serum triglycerides
in male albino rats
106
(24-b)
Effect of STZ-induced type 2 diabetes on serum total
cholesterol in male albino rats
106
(24-c)
Effect of STZ-induced type 2 diabetes on serum HDL-C in
male albino rats
106
(24-d)
Effect of STZ-induced type 2 diabetes on serum LDL-C in
male albino rats
106
(24-e)
Effect of STZ-induced type 2 diabetes on serum free fatty
acids in male albino rats
107
(24-f)
Effect of STZ-induced type 2 diabetes on non- HDL-C in
male albino rats
107
(24-g)
Effect of STZ-induced type 2 diabetes on TGs/HDL ratio in
male albino rats
107
(25)
Effect of STZ-induced type 2 diabetes on hepatic
malondialdehyde in male albino rats
109
(26)
Effect of STZ-induced type 2 diabetes on hepatic reduced
glutathione in male albino rats
109
(27)
Effect of STZ-induced type 2 diabetes on serum monocyte
chemoattractant protein-1 in male albino rats
111
(28)
Effect of STZ-induced type 2 diabetes on serum C-reactive
protein in male albino rats
111
(29)
Effect of STZ-induced type 2 diabetes on serum nitric oxide
in male albino rats
111
(30)
Effect of treatment with the studied drugs for 2 weeks on
fasting serum glucose in male albino rats
114
(31)
Effect of treatment with the studied drugs for 2 weeks on
fasting serum insulin in male albino rats
117
(32)
Effect of treatment with the studied drugs for 2 weeks on
HOMA-IR in male albino rats
120
(33-a)
Effect of treatment with the studied drugs for 2 weeks on
serum triglycerides in male albino rats
123
(33-b)
Effect of treatment with the studied drugs for 2 weeks on
serum total cholesterol in male albino rats
126
vi
Figure
Page
(33-c)
Effect of treatment with the studied drugs for 2 weeks on
serum high density lipoprotein cholesterol in male albino rats
129
(33-d)
Effect of treatment with the studied drugs for 2 weeks on
serum low density lipoprotein cholesterol in male albino rats
132
(33-e)
Effect of treatment with the studied drugs for 2 weeks on
serum free fatty acids in male albino rats
135
(33-f)
Effect of treatment with the studied drugs for 2 weeks on non-
HDL-cholesterol in male albino rats
138
(33-g)
Effect of treatment with the studied drugs for 2 weeks on
triglycerides to HDL-cholesterol ratio in male albino rats
141
(34)
Effect of treatment with the studied drugs for 2 weeks on
hepatic malondialdehyde in male albino rats
144
(35)
Effect of treatment with the studied drugs for 2 weeks on
hepatic reduced glutathione in male albino rats
147
(36)
Effect of treatment with the studied drugs for 2 weeks on
serum monocyte chemoattractant protein-1 in male albino rats
150
(37)
Effect of treatment with the studied drugs for 2 weeks on
serum C-reactive protein in male albino rats
153
(38)
Effect of treatment with the studied drugs for 2 weeks on
serum nitric oxide in male albino rats
156
(39)
Histopathological evaluation of pancreatic sections stained
with hematoxylin and eosin (H&E) stain (X 10).
158
(40)
Comparison of mean percentage change in biochemical
metabolic, oxidative stress and inflammatory parameters
between untreated and treated (metformin, L-cysteine and
their combination) experimentally induced type 2 diabetic
adult male rats
159
(41)
Comparison of mean percentage change in lipid profile
between untreated and treated (metformin, L-cysteine and
their combination) experimentally induced type 2 diabetic
adult male rats
160
vii
LIST OF ABBREVIATIONS
8-OH-Guanine :
8-hydroxy Guanine
ACC :
Acetyl-CoA carboxylase
ACEI :
Angiotensin converting enzyme inhibitors
ACOD :
Acyl-CoA oxidase
Acyl CS
:
Acyl CoA synthetase
ADP :
Adenosine diphosphate
AGEs :
Advanced glycation endproducts
AIDS :
Acquired immunodeficiency syndrome
Akt :
Apoptosis serine/therionine kinase
AMP : Adenosine monophosphate
AMPK :
Adenosine
monophosphate-activated
protein
kinase
ARB :
Angiotensin receptor blocker
ATP :
Adenosine triphosphate
ATPase :
Adenosinine triphosphatase enzyme
BH
4
:
Tetrahydrobiopterin cofactor
Ca
2+
: Calcium ion
CAT :
Catalase
Ccl2 :
Chemokine ligand 2
CCl
4
:
Carbon tetrachloride
Ccr2 :
Cognate receptor chemokine receptor 2
CD4 :
Cluster of differentiation 4
cNOS : Constitutive nitric oxide synthase
CoA :
Coenzyme A
CoQ
10
:
Coenzyme Q
10
CRP :
C-reactive protein
Cu :
Cupper
Cu/Zn SOD
:
Cupper zinc superoxide dismutase
DAG :
Diacylglycerol
DHAP
: dihydroxyacetone phosphate
DM :
Diabetes mellitus
viii
DNA :
Deoxyribonucleic acid
DPP-4 :
Dipeptidyl peptidase-4
DTNB :
5,5'-Dithiobis-2-nitrobenzoic acid
ECS :
Endocannabinoid system
EDTA
:
Disodium salt of ethylene diamine tetraacetic acid
ELISA :
Enzyme linked immunosorbent assay
eNOS :
Endothelial nitric oxide synthase
ESRF :
End stage renal failure
ETC :
Electron transport chain
FADH
2
:
Reduced flavin-adenine dinucleotide
FAS :
Fatty acid synthase
FBPase :
Fructose 1,6-bisphosphatase
FDA :
Food and drug administration
Fe :
Iron
FFAs :
Free fatty acids
FSG :
Fasting serum glucose
FSI :
Fasting serum insulin
GADA :
Glutamic acid decarboxylase autoantibodies
GAPDH :
Glyceraldehyde-3-phosphate dehydrogenase
GDM :
Gestational diabetes mellitus
GFAT : Glutamine:fructose-6-phosphate amidotransferase
GIP :
Gastric inhibitory polypeptide
Gln : Glutamine
GLP-1 :
Glucagon-like peptide-1
Glu
: Glutamate
GLUT1 :
Glucose transporter-1
GLUT2 :
Glucose transporter-2
GLUT3 :
Glucose transporter-3
GLUT4 :
Glucose transporter-4
GPx :
Glutathione peroxidase
GSH :
Reduced glutathione
GSSG :
Oxidized glutathione
GST :
Glutathione transferase
ix
H
2
O
: Water
molecule
H
2
O
2
:
Hydrogen peroxide
HBA
1C
:
Glycated Hemoglobin A
1C
HCl :
Hydrochloric acid
HDL-C :
High density lipoprotein cholesterol
HFD :
High fat diet
HIV-1 :
Human immunodeficiency virus-1
HLA :
Human leukocyte antigen
HMG-CoA :
3-hydroxy-3-methyl-glutaryl-Coenzyme A
HNF-1 :
Hepatic nuclear factor-1
HNF-4 :
Hepatic nuclear factor-4
HNO
2
: Nitrous oxide
HOCL :
Hypochlorous acid
HOMA-IR :
Homeostasis model assessment of insulin
resistance
HRO
2
·
: Hydroperoxyl
HRP :
Horseradish peroxidase enzyme
IAAs :
Insulin autoantibodies
ICAM-1 :
Intercellular adhesion molecule-1
ICAs :
Islet-cell autoantibodies
IDL :
Intermediate density lipoprotein
IGT :
Impaired glucose tolerance
IKK :
Inhibitor of nuclear factor- B kinase beta
IL-1 :
Interleukin-1
IL-10 : Interleukin-10
IL-2 :
Interleukin-2
IL-6 : Interleukin-6
IL-8 : Interleukin-8
iNOS :
Inducible nitric oxide synthase
IPF-1 :
Insulin promoter factor-1
IR :
Insulin resistance
IRS-1 :
Insulin receptor substrate-1
K
+
/Na
+
tartarate :
Potassium sodium tartarate
x
K
+
ATP channels :
Potassium channels adenosine triphosphate
KCl :
Potassium chloride
LADA :
Latent autoimmune diabetes in adults
LDL-C :
Low density lipoprotein cholesterol
LPL
: Lipoprotein lipase
LSD :
Least significant difference
MAOIs :
Monoamine oxidase inhibitors
MCP-1
:
Monocyte chemoattractant protein-1
MDA :
Malondialdehyde
microRNA : micro-ribonucleic acid
Mn-SOD :
Manganese superoxide dismutase
MODY :
Maturity onset diabetes of the young
mRNA :
Messenger ribonucleic acid
NAC : N-acetyl cysteine
NAD
+
:
Oxidized nicotinamide-adenine dinucleotide
NADH :
Reduced nicotinanide adenine dinucleotide
NADP : Oxidized Nicotinamide adenine dinucleotide
phosphate
NADPH : Reduced Nicotinamide adenine dinucleotide
phosphate
NaOH :
Sodium hydroxide
NED :
N-(1-naphthyl) ethylenediamine
NEFA :
Non-esterified fatty acids
NF- B
:
Nuclear factor kappa B
nNOS :
Neural nitric oxide synthase
NO :
Nitric oxide
NO
2
-
: Nitrite
NO
2
·-
:
Nitrogen dioxide
NO
3
-
: Nitrate
Non-HDL-C :
Non-high density lipoprotein cholesterol
NPH :
Neutral protamine
NSAIDs : Non steroidal anti-inflammatory drugs
O
2
:
Oxygen molecule
xi
O
2
-
:
Superoxide radical
OCT1 :
Organic cation transporter-1
OH :
Hydroxyl radical
ONOO
-
: Peroxynitre
P :
Phosphate
PAI-1 :
Plasminogen-activator inhibitor -1
PCOS :
Polycystic ovarian syndrome
PDX-1 :
Pancreas duodenum homeobox-1
PEPCK :
Phosphenolpyruvate carboxykinase
PI3K :
Phosphatidylinositol 3-kinase
PKC :
Protein kinase C
PPAR- :
Peroxisome proliferator-activated receptor alpha
PPAR- :
Peroxisome proliferator-activated receptor
gamma
Prx :
Peroxiredoxin
PUFAs :
Polyunsaturated fatty acids
RAGE :
Receptor for advanced glycation endproducts
rDNA :
Recombinant deoxyribonucleic acid
RNS :
Reactive nitrogen species
RO
2
:
Proxyl radical
RONOO :
Alkyl peroxynitrates
ROS :
Reactive oxygen species
rpm :
Rotation per minute
SDS :
Sodecyl sulphate
SGLT2 :
Sodium-glucose cotransporter-2
SH :
Thiol or sulfhydryl group
SOD :
Superoxide dismutase
SREBP :
Sterol regulatory element-binding protein
STZ : Streptozotocin
SUR1 :
Sulfonylurea receptor 1
SUs :
Sulfonylureas
T1DM :
Type 1 diabetes mellitus
T2DM :
Type 2 diabetes mellitus
xii
TBA :
Thiobarbituric acid
TBARS :
Thiobarbituric acid reactive substances
TBHB :
2,4,4-tribromo-3-hydroxy-benzoic acid
TC :
Total cholesterol
TCA :
Trichloroacetic acid
TGs :
Triglycerides
TMB :
3,3',5,5' tetramethylbenzidine
TMP :
1,1, 3,3-tetramethoxypropane
TNB
: 5-thionitrobenzoic
acid
TNF- :
Tumor necrosis factor-
Trx :
Thioredoxin
TxA2 :
Thromboxane A2
Tyr :
Tyrosine
TZDs :
Thiazolidinediones
UDP-GlcNac : Uridine diphospho-N-acetylglucosamine
VCAM-1
:
Vascular cell adhesion molecule-1
VEGF :
Vascular endothelial growth factor
VLDL :
Very low density lipoproteins
WHO :
World Health Organization
ZDF : Zucker diabetic fatty
Zn :
Zinc
BACKGROUND OF THE STUDY
Diabetes mellitus (DM) is a chronic multisystem disorder with
biochemical consequences and serious complications that affect many
organs. There are complex interactions between genetic, epigenetic,
environmental and behavioural factors that contribute to the development
of diabetes. Non-pharmacological and pharmacological interventions have
been used for diabetic management. Over the past few years, research has
started to focus on the use of novel adjuvant drugs as antioxidants and anti-
inflammatory drugs for better management, as it was revealed that both
oxidative stress and inflammation play a critical role in the disease
pathogenesis.
Metformin is a widely used oral antidiabetic agent for the
management of type 2 diabetes. Its primary mode of action appears to be
through improvement of insulin sensitivity and suppression of hepatic
gluconeogenesis and glycogenolysis. Moreover, it affects glucose transport
system, increases glucose utilization and delays its absorption from the
intestine. It also shows beneficial
effects on diabetes, as weight reduction
and improvements in lipid profile, inflammation and endothelial function.
L-cysteine is a semi-essential sulfur containing amino acid. One
important function of L-cysteine is that it is a precursor of glutathione,
which is pivotal for the detoxification of cellular oxidative stress. Dietary
intake of cysteine-rich proteins lowers the oxidative stress and insulin
resistance. It improves glycemic control, shows an anti-inflammatory effect
and implies a protective effect on pancreatic -cells.
Taking the above mentioned data in consideration, it seems that
combined therapy of metformin and an antioxidant like L-cysteine may be
of value in treatment of the diabetic state and amelioration of the oxidative
stress and inflammation associated with diabetes mellitus.
1
INTRODUCTION
Diabetes mellitus is the most common endocrine metabolic disorder,
affecting about 170 million people worldwide
(1)
. It represents a group of
diseases with complex heterogeneous etiology, characterized by chronic
hyperglycemia with carbohydrate, fat and protein metabolic
abnormalities
(2)
, which are due to insulin deficiency and/or insulin
resistance
(3)
. These abnormalities result in the impairment of uptake and
storage of glucose and reduced glucose utilization for energy purposes.
Defects in glucose metabolizing machinery and consistent efforts of the
physiological system to correct the imbalance in glucose metabolism place
an over-exertion on the endocrine system. Continuing deterioration of
endocrine control exacerbates the metabolic disturbances and leads
primarily to hyperglycemia
(4)
, then proceeds to the development of long-
term complications, such as microangiopathy; nephropathy, neuropathy and
retinopathy. The basis of these complications is a subject of great debate
and research. Hyperglycemia and metabolic derangement are accused as
the main causes of these long-standing changes in various organs.
Hyperglycemia may also lead to increased generation of free radicals and
reduced antioxidant defense system
(3)
.
Epidemiology of diabetes mellitus
Diabetes mellitus is a common growing disease, which is considered
epidemic by WHO. Its incidence in adults and adolescents have been
alarmingly rising in developed countries with estimate for an increase of
60% in the adult population above 30 years of age in 2025, with a higher
prevalence in the 45 to 64 years-old adults
(5)
. These increases are expected
because of population ageing and urbanization.
According to the WHO,
undiagnosed diabetes in Egypt will be about 8.8 million by the year 2025
(6)
.
2
Diabetes mellitus classification
The current classification includes four main categories
(7)
:
I- Type 1 diabetes, either type 1A (immune-mediated, e.g. latent
autoimmune diabetes in adults [LADA]) or type 1 B (idiopathic)
II- Type 2 diabetes
III- Other specific types
1.
Genetic defects of -cell function (maturity onset diabetes of the
young [MODY]). These defects may be in genes of hepatic nuclear
factor (HNF-1 or HNF-4 ) or insulin promoter factor-1 (IPF-1).
2.
Genetic defects in insulin action (Type A insulin resistance,
lipoatrophic diabetes).
3.
Diseases of the exocrine pancreas (pancreatitis, neoplasia, cystic
fibrosis, hemochromatosis).
4.
Endocrinopathies (acromegaly, Cushing's syndrome, glucagonoma,
pheochromocytoma, hyperthyroidism).
5.
Drug or chemical induced (vacor, streptozotocin, alloxan,
glucocorticoids, thyroid hormone, diazoxide, thiazide diuretics,
minoxidil, oral contraceptives, L-dopa).
6.
Infections (congenital rubella, cytomegalovirus).
7.
Uncommon forms of immune-mediated diabetes ("Stiff-man"
syndrome, anti-insulin receptor antibodies).
8.
Other genetic syndromes sometimes associated with diabetes (Down
syndrome, Klinefelter syndrome, Turner syndrome).
IV-Gestational diabetes mellitus
Gestational diabetes mellitus (GDM) is defined as any abnormal
carbohydrate intolerance that begins or is first recognized during
pregnancy
(8)
. It is associated with an increased risk of perinatal mortality
and congenital abnormalities, which is further increased by impaired
glycemic control
(9)
. It occurs in approximately 7% of all pregnancies and if
occurred once, it is likely to occur in subsequent pregnancies. Up to 70% of
women with GDM have a potential risk of developing type 2 diabetes
mellitus. The risk factors for developing gestational diabetes are similar to
3
those for type 2 diabetes, including family history, age, obesity and
ethnicity
(10)
. It is known that pregnancy is a diabetogenic state,
characterized by impaired insulin sensitivity, particularly in the second and
third trimester. This is due to changes in some hormones such as human
placental lactogen, progesterone, prolactin and cortisol that antagonize the
effects
of
insulin
and
decrease phosphorylation of insulin receptor
substrate-1 (IRS-1), triggering a state of insulin resistance. Logically, the
pancreas should compensate for this demand by increasing insulin
secretion. However in GDM, there is deterioration of beta cell function,
particularly the first phase insulin secretion
(8)
.
An intermediate group of individuals with impaired fasting glucose
and/or impaired glucose tolerance was classified as "pre-diabetics". Their
progression to diabetes is common, particularly when non-pharmacological
interventions, such as lifestyle changes are not provided
(7)
.
I-Type 1 diabetes mellitus
Type 1 diabetes mellitus (T1DM) is an organ-specific progressive
cellular-mediated autoimmune disease characterized by a defect in insulin
production, as a result of selective and massive destruction of islet -cells
(8090%). It accounts for only about 510% of all cases of diabetes;
however, its incidence continues to increase worldwide. The progression of
the autoimmune process is generally slow and may take several years
before the onset of the clinical diabetes
(11)
. Markers of the immune -cell
destruction, including circulating insulin autoantibodies (IAAs), islet-cell
autoantibodies (ICAs) and glutamic acid decarboxylase autoantibodies
(GADA), are present in 90% of patients at the time of diagnosis
(7)
.
Two forms are identified:
· Type 1A DM, which results from a cell-mediated autoimmune attack
on -cells
and has a strong genetic component, inherited through the
human leukocyte antigen (HLA) complex, mainly HLA-DR3 and
HLA-DR4
(12)
, but the factors that trigger onset of clinical disease
remain largely unknown.
· Type 1B DM (idiopathic) is a far less frequent form, has no known
cause, and occurs mostly in individuals of Asian or African
4
descent
(7)
. This form of diabetes lacks evidence for -cell
autoimmunity and is not HLA associated. While the disease is often
manifested by severe insulinopenia and/or ketoacidosis, -cell
function often recovers, rendering almost normal glucose levels
(13)
.
Starting generally at a young age, T1DM is also referred to as
`juvenile diabetes'. Indeed, it affects children and young adults in particular
and generally occurs before the age of 40, with incidence peaks at 2, 4-6
and 10-14 years
(11)
. However, it can also occur at any age, even as late as
in the eighth and ninth decades of life. The slow rate of -cell destruction in
adults may mask the presentation, making it difficult to distinguish from
type 2 diabetes. This type of diabetes is known as "Latent Autoimmune
Diabetes in Adults"
(13)
.
Patients with type 1 diabetes are severely insulin deficient and are
dependent on insulin replacement therapy for their survival
(13)
.
II-Type 2 diabetes mellitus
Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder of
polygenic nature, that is characterized by defects in both insulin action and
insulin secretion
(14)
. T2DM affects nowadays more than 150 million people
worldwide and is projected to increase to 439 million worldwide in
2030
(15)
. By the end of the 20
th
century, its incidence has increased
dramatically in children and adolescents, as a result of the rise in childhood
obesity and is now continuing to rise, changing the demographics of the
disease in this group
(16)
.
Genetic, epigenetic and environmental factors have been implicated in
type 2 diabetes mellitus pathogenesis with increasing evidences that
epigenetic factors play a key role in the complex interplay between
them
(17)
. Epigenetic mechanisms are commonly associated to gene
silencing and transcriptional regulation of genes
(18)
. The epigenetic control
of gene expression is based on modulation of chromatin structure and
accessibility to transcription factors, which is achieved by multiple
mechanisms. These mechanisms involve methylationdemethylation of
cytidineguanosine sequences in the promoter regions, acetylation
deacetylation of lysine residues of core histones in the nucleosome and
5
presence of microRNA molecules, which bind to their complementary
sequences in the 3 end of mRNA and reduce the rate of protein
synthesis
(19)
. Actions of major pathological mediators of diabetes and its
complications such as hyperglycemia, oxidative stress and inflammation
can lead to the dysregulation of these epigenetic mechanisms
(17)
.
Insulin resistance in peripheral tissues, such as muscle and fat, is often
the earliest recognizable feature of T2DM, results in a compensatory
hyperinsulinemia that promotes further weight gain. This occurs until the -
cells can no longer compensate for the increased insulin resistance, then -
cell failure and hyperglycemia ensue
(20)
. It is also associated with co-
morbidities, such as hypertension, hyperlipidemia and cardiovascular
diseases, which taken together comprise the `Metabolic Syndrome'
(20)
.
Similar to adults, obesity in children appears to be a major risk factor
for type 2 diabetes
(21)
. Many studies show a strong family history among
affected youth, with 45-80% having at least one parent with diabetes and
74-100% having a first- or second-degree relative with type 2 diabetes
(22)
.
Until now, type 2 diabetes was typically regarded as a disease of the
middle-aged and elderly. While it is still true that this age group maintains
a higher risk than the younger adults do, evidence is accumulating that
onset in children and adolescents is increasingly common. Onset of
diabetes in childhood or adolescence, around the time of puberty, heralds
many years of disease and increases the risk of occurrence of the full range
of both micro- and macrovascular complications
(23)
.
Although a mother could transmit genetic susceptibility to her
offspring, it is more likely that maternal diabetes increases the risk of
diabetes in children by altering the intrauterine environment, which can
impair the normal -cell development and function as well as the insulin
sensitivity of skeletal muscle
(24)
. Moreover, malnutrition during fetal or
early life and low birth weight appear to be associated with an increased
risk of adulthood insulin resistance, glucose intolerance, T2DM,
dyslipidemia and hypertension
(25)
.
6
Normal glucose homeostasis
Maintenance of serum glucose concentrations within a normal
physiological range (fasting blood glucose level 70-110 mg/dl), is primarily
accomplished by two pancreatic hormones; insulin, secreted by the -cells
and glucagon, secreted by -cells
(26)
. Derangements of glucagon or insulin
regulation can result in hyperglycemia or hypoglycemia, respectively.
In the postabsorptive state, the majority of total body glucose disposal
takes place in insulin-independent tissues
(27)
. Approximately 50% of all
glucose utilization occurs in the brain and 25% of glucose uptake occurs in
the splanchnic area (liver and the gastrointestinal tissue). The remaining
25% of glucose metabolism takes place in insulin-dependent tissues,
primarily muscle with only a small amount being metabolized by
adipocytes
(28)
. Approximately 85% of endogenous glucose production is
derived from the liver and the remaining amount is produced by the kidney.
Glycogenolysis and gluconeogenesis contribute equally to the basal rate of
hepatic glucose production
(27)
.
In the postprandial state, the maintenance of whole-body glucose
homeostasis is dependent upon a normal insulin secretory response and
normal tissue sensitivity to the effects of hyperinsulinemia and
hyperglycemia to augment glucose disposal. This occurs by three tightly
coupled mechanisms: (i) suppression of endogenous glucose production and
increase glycogen synthesis; (ii) stimulation of glucose uptake by the
splanchnic tissues; and (iii) stimulation of glucose uptake by peripheral
tissues, primarily muscle
(27)
.
The route of glucose administration also plays an important role in the
overall glucose homeostasis. Oral glucose ingestion has a potentiating
effect on insulin secretion that the insulin concentrations in the circulation
increase very rapidly (by at least two to threefold) after oral glucose, when
compared to a similar intravenous bolus of glucose. This potentiating effect
of oral glucose administration is known as "the incretin effect" and is
related to the release of glucagon-like peptide-1 (GLP-1) and glucose-
dependent insulinotropic polypeptide (also called gastric inhibitory
polypeptide) (GIP) from the gastrointestinal tissues
(29)
.
7
Glucose homeostasis in type 2 diabetes mellitus
Type 2 diabetic individuals are characterized by defects in insulin
secretion; insulin resistance involving muscle, liver and the adipocytes; and
abnormalities in splanchnic glucose uptake
(28)
.
Insulin secretion in insulin resistant, non-diabetic individuals is
increased in proportion to the severity of the insulin resistance and glucose
tolerance remains normal. Thus, their pancreas is able to "read" the severity
of insulin resistance and adjust its secretion of insulin. However, the
progression to type 2 diabetes with mild fasting hyperglycemia (120-140
mg/dl) is heralded by an inability of the -cell to maintain its previously high
rate of insulin secretion in response to a glucose challenge, without any
further or only minimal deterioration in tissue sensitivity to insulin
(27)
.
The relationship between the fasting plasma glucose concentration and
the fasting plasma insulin concentration resembles an inverted U or
horseshoe. As the fasting plasma glucose concentration rises from 80 to
140 mg/dl, the fasting plasma insulin concentration increases progressively,
peaking at a value that is 2-2.5 folds greater than in normal weight, non-
diabetic, age-matched controls. The progressive rise in fasting plasma
insulin level can be viewed as an adaptive response of the pancreas to
offset the progressive deterioration in glucose homeostasis. However, when
the fasting plasma glucose concentration exceeds 140 mg/dl, the beta cell is
unable to maintain its elevated rate of insulin secretion and the fasting
insulin concentration declines precipitously. This decrease in fasting insulin
level has important physiologic implications, since at this point; hepatic
glucose production begins to rise, which is correlated with the severity of
fasting and postprandial hyperglycemia
(27)
.
Moreover, the largest part of the impairment in insulin-mediated
glucose uptake is accounted for a defect in muscle glucose disposal. Thus
in the basal state, the liver represents a major site of insulin resistance
(30)
;
however in the postprandial state, both decreased muscle glucose uptake
and impaired suppression of hepatic glucose production contribute to the
insulin resistance, together with defect in insulin secretion, are the causes
of postprandial hyperglycemia. It should be noted that brain glucose uptake
occurs at the same rate during absorptive and postabsorptive periods and is
not altered in type 2 diabetes
(28)
.
8
Pathophysiology of type 2 diabetes mellitus
Insulin resistance:
Insulin resistance (IR) is the impaired sensitivity and attenuated
response to insulin in its main target organs, adipose tissue, liver, and
muscle, leading to compensatory hyperinsulinemia
(31)
.
In adipose tissue, insulin decreases lipolysis, thereby reducing FFAs
efflux from the adipocytes. However, intra-abdominal fat is metabolically
distinct from subcutaneous fat, as it is more lipolytically active and less
sensitive to the antilipolytic effects of insulin. In liver, insulin inhibits
gluconeogenesis by reducing key enzyme activities. While in skeletal
muscle, insulin predominantly induces glucose uptake by stimulating the
translocation of the GLUT4 glucose transporter to the plasma membrane
and promotes glycogen synthesis
(32)
.
Insulin resistance leads to increased lipolysis of the stored
triacylglycerol molecules with subsequent increase circulating FFAs
concentrations and ectopic fat accumulation. This results in increases in the
flux of FFAs from fat to liver and periphery. Excess delivery of FFAs
stimulates liver glucose and triglycerides production
(33)
, impedes insulin
mediated glucose uptake and decreases glycogen synthesis in skeletal
muscle
(34)
, as well as impairs vascular reactivity and induces
inflammation
(35)
. This is shown in Figure (1).
At the molecular level, impaired insulin signaling results from reduced
receptor expression or mutations or post-translational modifications of the
insulin receptor itself or any of its downstream effector molecules. These
reduce tyrosine-specific protein kinase activity or its ability to
phosphorylate substrate proteins (IRS)
(36)
resulting in phosphatidylinositol-
3-kinase (PI3K)/Akt pathway impairment
(37)
.
To date, several methods for evaluating insulin resistance in humans
have been reported such as fasting serum insulin levels, homeostasis model
assessment of insulin resistance (HOMA-IR) and insulin tolerance test
(38)
.
Because abnormalities in insulin action are poorly detected by a single
determination of either glucose or insulin levels, the insulin resistance is
commonly evaluated by HOMA-IR, which is likely to be the most simple
and repeatable index
(38, 39)
.
9
Glucotoxicity:
Insulin resistance results in chronic fasting and postprandial
hyperglycemia. Chronic hyperglycemia may deplete insulin secretory
granules from -cells, leaving less insulin ready for release in response to a
new glucose stimulus
(40)
. This has led to the concept of glucose toxicity,
which implies the development of irreversible damage to cellular
components of insulin production over time. Hyperglycemia stimulates the
production of large amounts of reactive oxygen species (ROS) in -cells.
Due to ROS interference, loss of pancreas duodenum homeobox-1 (PDX-1)
has been proposed as an important mechanism leading to -cell
dysfunction. PDX-1 is a necessary transcription factor for insulin gene
expression and glucose-induced insulin secretion, besides being a critical
regulator of -cell survival. Additionally, ROS are known to enhance NF-
B activity, which potentially induces -cell apoptosis
(41)
.
Lipotoxicity:
Chronically elevated free fatty acids (FFAs) level results from the
resistance to the antilipolytic effect of insulin. It is known that FFAs
acutely stimulate insulin secretion, but chronically impair insulin secretion,
induce further hepatic and muscle insulin resistance, stimulate
gluconeogenesis and cause a decrease -cell function and mass, an effect
referred to as -cell lipotoxicity
(42)
.
In the presence of glucose, fatty acid oxidation in -cells is inhibited
and accumulation of long-chain acyl coenzyme A occurs. This mechanism
has been proposed to be an integral part of the normal insulin secretory
process. However, its excessive accumulation can diminish the insulin
secretory process by opening -cell potassium channels. Another
mechanism might involve apoptosis of -cells, possibly via generation of
nitric oxide through inducible nitric oxide synthase (iNOS) activation
which results in great production of toxic peroxynitrite (ONOO
-
)
(43)
.
Thus, glucolipotoxicity may play an important role in the pathogenesis
of hyperglycemia and dyslipidemia associated with type 2 diabetes
(44)
.
10
Dys
`tria
incre
chol
lipop
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Figu
slipidem
Diabetic
ad' profile
ease in p
lesterol an
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(4
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ure (1): M
fu
ty
mia:
dyslipide
e, known
plasma tri
nd a conco
45, 46)
. Thes
erosclerosis
Model for
function/m
ype 2 diab
emia is ty
as athero
iglycerides
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se lipid ab
s and card
r the eff
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betes
(47)
.
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ogenic dy
s, a decr
ncrease in
bnormaliti
diovascular
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defined by
yslipidemia
rease in h
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ed low-de
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lipid
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(46)
.
-cell
is of
11
In diabetic patients, there is typically a preponderance of smaller,
denser, oxidized LDL particles, which may increase atherogenicity and
cardiovascular risk, even if the absolute concentration of LDL cholesterol
is not elevated
(45)
.
Non-HDL cholesterol (non-HDL-C) is a new measure that reflects the
combined lipid profile change. It encompasses all cholesterol present in the
potentially atherogenic lipoprotein particles (VLDL, remnants, IDL and
LDL). Non-HDL-C has been shown to correlate with coronary artery
disease severity and progression, as well as predicts cardiovascular
morbidity and mortality in patients with diabetes
(48)
.
Another simple tool, Triglycerides to HDL-cholesterol ratio (TGs:
HDL-C) has been proposed as an atherogenic index, that has proven to be a
highly significant predictor of myocardial infarction, even stronger than
total cholesterol to HDL-C ratio and LDL-C to HDL-C ratio
(49)
. Moreover,
a significant negative relationship between TGs: HDL-C ratio and insulin
sensitivity was observed. Thus a TGs: HDL-C ratio > 3.5 provides a simple
mean of identifying insulin resistant, dyslipidemic patients who are at
increased risk of cardiovascular diseases
(50)
.
The precise pathogenesis of diabetic dyslipidemia is not fully known;
nevertheless, a large body of evidence suggests that insulin resistance has a
central role in the development of this condition as a result of the increased
influx of free fatty acids from insulin-resistant fat cells into the liver, in the
presence of adequate glycogen stores
(51)
.
Diabetic complications and their pathogenesis
Hyperosmolar hyperglycemic non-ketotic state
It is one of the major acute complications, which is a life-threatening
condition, commonly occurs in elderly patients with type 2 diabetes. There
is almost always a precipitating factor which include; the use of some
drugs, acute situations and chronic diseases. Abnormal thirst sensation and
limited access to water also facilitate development of this syndrome. It is
associated with four major clinical features, which are severe
hyperglycemia (blood glucose more than 600 mg/dl), absent or slight
12
ketosis, plasma hyperosmolarity and profound dehydration. Treatment of
this state should be started immediately with the determination and
correction of the precipitating event and lifesaving measures, while the
other clinical manifestations should be corrected with the use of
appropriate fluids and insulin
(52)
.
However, chronic complications can be divided into microvascular;
affecting eyes, kidneys and nerves and macrovascular; affecting the
coronary, cerebral and peripheral vascular systems
(53)
.
Microvascular complications
In fact, microvascular complications can begin in developing at least 7
years before the clinical diagnosis of type 2 diabetes. Conversely; type 1
patients may not develop signs of microvascular complications until 10
years after diagnosis of diabetes
(54)
.
Nephropathy: Diabetic nephropathy is a frequent complication of
type 1 and type 2 diabetes mellitus, characterized by excessive urinary
albumin excretion, hypertension and progressive renal insufficiency. The
natural history of diabetic nephropathy has 5 stages; which include
hyperfiltration with normal renal function; histological changes without
clinically evident disease; incipient diabetic nephropathy or
microalbuminuria; overt diabetic nephropathy (macroalbuminuria and
reduced renal function); and renal failure requiring dialysis (end stage renal
disease)
(55)
.
Neuropathy: Diabetic peripheral neuropathy is one of the most
prevalent and complicated conditions to manage among diabetic patients.
Diabetes is the major contributing reason for non-traumatic lower extremity
amputations (more than 60% of cases). Ischemia occurs because of
compromised vasculature that fails to deliver oxygen and nutrients to nerve
fibers. This results in damage to myelin sheath covering and insulating
nerve.
The most common form involves the somatic nervous system;
however, the autonomic nervous system may be affected in some patients.
Sensorimotor neuropathy is characterized by symptoms; such as burning,
tingling sensations and allodynia. Autonomic neuropathy can cause
gastroparesis, sexual dysfunction and bladder incontinence
(54)
.
13
Retinopathy: Diabetic retinopathy is the most frequent cause of new
cases of blindness among adults aged 20-74 years
(56)
. Non-proliferative
retinopathy produces blood vessel changes within the retina, which include
weakened blood vessel walls, leakage of fluids and loss of circulation. It
generally does not interfere with vision
(54)
. However, if left untreated, it
can progress to proliferative retinopathy, that is very serious and severe.
It occurs when new blood vessels branch out or proliferate in and around
the retina
(56)
. It can cause bleeding into the fluid-filled center of the eye or
swelling of the retina, leading to blindness. The duration of diabetes and
the degree of hyperglycemia are probably the strongest predictors for
development and progression of retinopathy
(54)
.
Macrovascular complications
The hallmark of diabetic macrovascular disease is the accelerated
atherosclerosis; involving the aorta and the large and medium-sized
arteries, which is a leading cause of morbidity and mortality in diabetes
(54)
.
Accelerated atherosclerosis caused by accumulation of lipoproteins within
the vessel wall, resulting in the increased formation of fibrous plaques
(53)
.
Hyperglycemia also affects endothelial function, resulting in increased
permeability, altered release of vasoactive substances, increased production
of procoagulation proteins and decreased production of fibrinolytic factors
(53)
. All these changes result in atherosclerotic heart disease, myocardial
infarction and sudden death; peripheral vascular disease and
cerebrovascular disease, including cerebral hemorrhage, infarction and
stroke.
Hyperglycemia causes tissue damage through four major mechanisms.
Several evidences indicate that all these mechanisms are activated by a
single upstream event, which is the mitochondrial overproduction of
reactive oxygen species
(57)
, Figure (2).
Increased polyol pathway flux
The polyol pathway is based on a family of aldo-keto reductase
enzymes, which can use as substrates a wide variety of carbonyl
compounds and reduce them by NADPH to their respective sugar alcohols
(polyols). Glucose is converted to sorbitol by the enzyme aldose reductase,
14
which is then oxidized to fructose by the enzyme sorbitol dehydrogenase,
using NAD
+
as a cofactor. Aldose reductase is found in tissues such as
nerve, retina, lens, glomerulus and vascular cells. In many of these tissues,
glucose uptake is mediated by insulin-independent GLUTs; intracellular
glucose concentrations, therefore, rise in parallel with hyperglycemia
(57)
.
Several mechanisms include sorbitol-induced osmotic stress,
increased cytosolic NADH/NAD
+
and decreased cytosolic NADPH have
been proposed to explain tissue damage resulted from this pathway
(58)
. The
most cited is an increase in redox stress, caused by the consumption of
NADPH, a cofactor required to regenerate reduced glutathione (GSH),
which is an important scavenger of ROS. This could induce or exacerbate
intracellular oxidative stress
(57)
.
Increased intracellular advanced glycation endproducts
(AGEs) formation and increased expression of the receptor
for AGEs (RAGE)
AGEs are formed by the non-enzymatic reaction of glucose and other
glycating compounds derived both from glucose and fatty acids with
proteins
(59)
. Intracellular production of AGE precursors can damage cells by
altering protein functions and binding of plasma proteins modified by AGE
precursors to RAGE on cells such as macrophages and vascular endothelial
cells. RAGE binding induces the production of ROS, which in turn activates
the pleiotropic transcription factor nuclear factor (NF- B), causing multiple
pathological changes in gene expression
(60)
. These effects induce
procoagulatory changes and increase the adhesion of inflammatory cells to
the endothelium. In addition, this binding appears to mediate, in part, the
increased vascular permeability induced by diabetes, probably through the
induction of VEGF
(57)
.
Increased protein kinase C (PKC) activation
PKC activation results primarily from enhanced de-novo synthesis of
diacylglycerol (DAG) from glucose via triose phosphate. Evidence
suggests that the enhanced activity of PKC isoforms could also result from
the interaction between AGEs and their cell-surface receptors
(57)
. PKC
activation implicated in many processes; such as increased vascular
15
permeability, angiogenesis, blood flow abnormalities, capillary and
vascular occlusion, which are involved in the pathology of diabetic
complications
(58)
.
Increased hexosamine pathway flux
Hyperglycemia and elevated free fatty acids also appear to contribute
to the pathogenesis of diabetic complications by increasing the flux of
glucose and fructose-6-phophate into the hexosamine pathway, leading to
increases in the transcription of some key genes and alteration in protein
functions such as eNOS inhibition
(57)
.
Mitochondrial superoxide overproduction
It has now been established that all of the different pathogenic
mechanisms described above stem from a single hyperglycemia-induced
process, namely overproduction of superoxide by the mitochondrial
electron-transport chain that can damage cells in numerous ways. It is
hypothesized that excess ROS inhibits GAPDH (glyceraldehyde-3-
phosphate dehydrogenase), a glycolytic key enzyme promoting shunting of
upstream glucose metabolites into the aforementioned pathways
(57)
. This
overproduction of ROS can be prevented by manganese superoxide
dismutase
(61)
.
16
Figu
ure (2): M
m
g
Mitochond
major pat
glyceralde
drial over
thways
ehyde-3-ph
rproductio
of hyperg
hosphate d
on of sup
glycemic
dehydroge
peroxide
damage
enase (GA
activates
by inhib
APDH)
(62)
the
biting
.
17
Obesity, inflammation and insulin resistance
Although genetic predisposition to insulin resistance exists, it is
widely accepted that the increasingly sedentary lifestyle; such as
consumption of a high caloric diet and lack of exercise, have increased the
global prevalence of not only insulin resistance and diabetes, but also of
obesity
(63)
. Between 60% and 90% of cases of type 2 diabetes now appear
to be related to obesity
(64)
. The close association of these two common
metabolic disorders has been referred to as "diabesity"
(65)
. Adipocytes are
not merely a site for storage of energy in the form of triglycerides, but also
a source of many adipokines
(63)
that have effects on many peripheral
tissues, including skeletal muscles and liver. As body weight increases,
there is expansion of the adipose tissue mass, particularly visceral intra-
abdominal adipose tissue, resulting in, not only excessive free fatty acids
release, but also altered release of these adipokines
(66)
. Increased release of
various inflammatory cytokines, such as tumor necrosis factor- (TNF- ),
IL-6, MCP-1 and resistin; mainly from visceral fat and leptin; mainly from
subcutaneous fat, together with decreased release of adiponectin contribute
to the whole body insulin resistance
(67)
. Figure (3) shows how
inflammation contributes to develop insulin resistance and type 2 diabetes
mellitus.
The inflammation is triggered in the adipose tissue by macrophages,
which form ring-like structures surrounding dead adipocytes. As adipose
tissue expands during the development of obesity, certain regions become
hypoperfused, leading to adipocyte microhypoxia and cell death. Adipocyte
hypoxia and death trigger a series of proinflammatory program, which in
turn recruit new macrophages. Another proposed mechanism is the
activation of inflammatory pathway by oxidative stress. Hyperglycemia
and high fat diet have been shown to increase ROS production, via multiple
pathways; such as NADPH oxidase activation, which in turn activates
nuclear factor- B, triggering inflammatory response in adipose tissue
(68)
.
TNF- , resistin, IL-6 and other cytokines appear to participate in the
induction and maintenance of the chronic low-grade inflammatory state;
which is one of the hallmarks of obesity and type 2 diabetes
(69)
. IL-6 may
interfere with insulin signaling, inhibit lipoprotein lipase activity and
18
increase concentrations of non-esterified fatty acids (NEFA), contributing
to dyslipidemia and insulin resistance
(70)
. In addition, IL-6 stimulates the
secretion of further proinflammatory cytokines; such as IL-1 and increases
the hepatic production of CRP, thus explaining its increase in the metabolic
syndrome and diabetes
(71)
.
C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-
1) and other chemokines have essential roles in the recruitment and
activation of macrophages in the adipose tissue and in the initiation of
inflammation
(69, 72)
. CRP activation of monocytes increases the expression
of Ccr2, the receptor for MCP-1
(72)
. Overexpression of MCP-1 causes
inhibition of Akt and tyrosine phosphorylation in liver and skeletal muscle,
which contributes to insulin resistance
(73, 74)
. This demonstrates a clear
association between increased levels of MCP-1 and CRP with the
decreased insulin sensitivity and increased vascular inflammation
(75)
,
explaining the increased risk of atherosclerosis, cardiovascular disease and
stroke in diabetic patients
(76, 77)
.
Figu
ure (3): D
p
o
in
f
s
a
Developm
precedes th
obesity an
nflammat
for insulin
some poin
and diabet
ment of ty
he develop
nd is ind
ion in adi
n resistance
nt, -cell c
tes ensues
ype 2 d
pment of
duced by
ipose tissu
e by hype
compensat
(63)
.
diabetes;
hyperglyc
adipokine
ue. Pancre
rsecretion
tion is fol
insulin re
cemia is a
es, FFAs,
eatic -cel
n of insulin
llowed by
esistance
associated
, and chr
lls compen
n. Howeve
y -cell fa
19
that
with
ronic
nsate
er, at
ailure
20
This state of proatherogenesis and low-grade inflammation is known
to cause induction of inducible nitric oxide synthase (iNOS), increasing
nitric oxide production
(78)
. Nitric oxide (NO) is a free radical, known to act
as a biological messenger in mammals. It has a dual role as a mediator of
physiological and pathophysiological processes.
In pancreatic islets, excess NO is produced on exposure to cytokines,
which mediates -cell injury and leads to diabetes mellitus. Nitric oxide
can also combine with oxygen to produce potent cellular killers, such as the
highly toxic hydroxyl radical (OHÚ) and peroxynitrite (ONOO
-
). In diabetes
mellitus, there is increased breakdown of NO by superoxide, resulting in
the excessive formation of peroxynitrite, a potent oxidant that can attack
many types of biological molecules. High levels of peroxynitrite cause
initation of lipid peroxidation, sulfhydryl oxidation, nitration of some
amino acids, direct DNA damage and oxidation of antioxidants
(3)
.
Oxidative stress in diabetes mellitus
Oxidative stress refers to a situation of a serious imbalance between
free radical-generating and radical-scavenging systems; i.e. increased free
radical production or reduced activity of antioxidant defenses or both,
leading to potential tissue damage
(79)
. There is currently great interest in
the potential contribution of reactive oxygen species (ROS) in pathogenesis
of diabetes and more importantly in the development of secondary
complications of diabetes
(80)
.
Free radical species include a variety of highly reactive molecules,
such as ROS and reactive nitrogen species (RNS). ROS include free
radicals such as superoxide (O
2
·-
), hydroxyl (OH
·
), peroxyl (RO
2
·
),
hydroperoxyl (HRO
2
·-
), as well as non-radical species such as hydrogen
peroxide (H
2
O
2
) and hypochlorous acid (HOCl). RNS include free radicals
like nitric oxide (NO
·
) and nitrogen dioxide (NO
2
·-
), as well as non-radicals
such as peroxynitrite (ONOO
-
), nitrous oxide (HNO
2
) and alkyl
peroxynitrates (RONOO)
(81)
. Production of one ROS or RNS may lead to
the production of others through radical chain reactions
(82)
. Of these
reactive molecules; O
2
·-
, NO
·
and ONOO
-
are the most widely studied
species, as they play important roles in diabetic complications
(83)
.
21
To avoid free radical overproduction, antioxidants are synthesized to
neutralize free radicals. Antioxidants include a manifold of enzymes, such
as superoxide dismutase (SOD), catalase, glutathione peroxidase and
glutathione reductase, as well as many non-enzymatic antioxidants as
vitamin A, C and E
(84)
. This is shown in Figure (4).
Free radicals, at physiological levels, play a key role in defense
mechanisms as seen in phagocytosis and neutrophil function. They are also
involved in gene transcription and, to some extent, acts as signaling
molecules. However, excess generation of free radicals in oxidative stress
has pathological consequences, including damage to nucleic acid, proteins
and lipids, causing tissue injury and cell death
(83)
.
Oxidative damage to DNA, lipids and proteins
1- Nucleic acid:
The hydroxyl radical is known to react with all components of the
DNA molecule, damaging both the purine and pyrimidine bases and the
deoxyribose backbone, causing base degeneration, single strand breakage
and cross-linking to proteins. The most extensively studied DNA lesion is
the formation of 8-OH-Guanine. Permanent modification of genetic
material, resulting from these oxidative damage incidents, represents the
first step involved in mutagenesis, carcinogenesis and ageing
(85, 86)
.
2- Proteins:
Collectively, ROS can lead to oxidation of the side chain of amino
acids residues of proteins, particularly methionine and cysteine residues,
forming protein-protein cross-linkages and oxidation of the protein
backbone
(87)
, resulting in protein fragmentation, denaturation, inactivation,
altered electrical charge and increased susceptibility to proteolysis
(88)
. The
concentration of carbonyl groups is a good measure of ROS-mediated
protein oxidation
(89)
.
22
3- Membrane lipids:
ROS attack polyunsaturated fatty acids (PUFAs) of phospholipids in
the cell membranes, which are extremely sensitive to oxidation because of
double and single bonds arrangement
(90)
. The removal of a hydrogen atom
leaves behind an unpaired electron on the carbon atom to which it was
originally attached. The resulting carbon-centered lipid radical can have
several fates, but the most likely one is to undergo molecular
rearrangement, followed by reaction with O
2
to give a peroxyl radical,
which are capable of abstracting hydrogen from adjacent fatty acid side
chains and so propagating the chain reaction of lipid peroxidation. Hence, a
single initiation event can result in conversion of hundreds of fatty acid
side chains into lipid hydroperoxides
(91)
. Further decomposition of these
lipid hydroperoxides produces toxic aldehydes; in particular 4-
hydroxynonenal and malondialdehyde
(92)
.
The occurrence of lipid peroxidation in biological membranes causes
impairment of membrane functioning, changes in fluidity, inactivation of
membrane-bound receptors and enzymes, and increased non-specific
permeability to ions
(93)
. Thus, lipid peroxidation in-vivo has been
implicated as the underlying mechanisms in numerous disorders and
diseases, such as cardiovascular diseases, atherosclerosis, liver cirrhosis,
cancer, neurological disorders, diabetes mellitus, rheumatoid arthritis and
aging
(89)
.
Malondialdehyde (MDA) is a major highly toxic by-product formed
by PUFAs peroxidation. MDA can react both irreversibly and reversibly
with proteins, DNA and phospholipids, resulting in profound mutagenic
and carcinogenic effects
(92, 94)
. The determination of plasma, urine or other
tissue MDA concentrations using thiobarbituric acid (TBA reaction)
continues to be widely used as a marker of oxidative stress, as its level
correlates with the extent of lipid peroxidation
(95)
.
Figu
ure (4): T
a
The cellula
and antiox
ar origins
xidant sys
of reactiv
tems
(68, 83
ve oxygen
3)
.
species, th
heir target
23
ts
24
Sources of oxidative stress in diabetes
Multiple sources of oxidative stress in diabetes including enzymatic,
non-enzymatic and mitochondrial pathways have been reported
(81)
.
Enzymatic sources of augmented generation of reactive species in
diabetes include NOS, NAD(P)H oxidase and xanthine oxidase . If NOS
lacks its substrate L-arginine or one of its cofactors, NOS may produce O
2
·
-
instead of NO
·
and this is referred to as the uncoupled state of NOS.
NAD(P)H oxidase is a membrane associated enzyme that consists of five
subunits and is a major source of O
2
·
-
production
(81)
. There is plausible
evidence that protein kinase C (PKC), which is stimulated in diabetes via
multiple mechanisms, activates NAD(P)H oxidase
(82)
.
Non-enzymatic sources of oxidative stress originate from
hyperglycemia, which can directly increase ROS generation. Glucose can
undergo auto-oxidation and generate OH
·
radicals. In addition, glucose
reacts with proteins in a non-enzymatic manner, leading to the development
of Amadori products followed by formation of advanced glycation
endproducts (AGEs). ROS is generated at multiple steps during this process
(96)
. Once AGEs are formed, they bind to various receptors termed RAGE
and this step is also generating ROS
(97)
. Moreover, cellular hyperglycemia
in diabetes leads to the depletion of NADPH, through the polyol pathway,
resulting in enhanced production of O
2
·
í (98)
.
The mitochondrial respiratory chain is another source of non-
enzymatic generation of reactive species. During the oxidative
phosphorylation process, electrons are transferred from electron carriers
NADH and FADH
2
, through four complexes in the inner mitochondrial
membrane, to oxygen, generating ATP and O
2
·
í
, which is immediately
eliminated by natural defense
(83)
. However, in the diabetic cells, more
glucose is oxidized by Krebs cycle, which pushes more NADH and FADH
2
into the electron transport chain (ETC) thereby overwhelming complex III
of ETC, where the transfer of electrons is blocked. Thus, the generated
electrons are directly donated to molecular oxygen, one at a time,
generating excessive superoxide
(57)
. Therefore in diabetes, electron transfer
and oxidative phosphorylation are uncoupled, resulting in excessive O
2
·
í
formation and inefficient ATP synthesis
(99)
.
Antioxidants
25
Reactive oxygen species can be eliminated by a number of antioxidant
defense mechanisms, which involve both enzymatic and non-enzymatic
strategies. They work in synergy with each other and against different types
of free radicals
(100)
. Hyperglycemia not only engenders free radicals, but
also impairs the endogenous antioxidant defense system and causes
inflammation in many ways in diabetes mellitus
(101)
, Figure (5). Decreases
in the activities of SOD, catalase and glutathione peroxidase; decreased
levels of glutathione and elevated concentrations of thiobarbituric acid
reactants are consistently observed in diabetic patients and in
experimentally-induced diabetes
(100)
.
Figure (5): Schematic of the effects of chronic oxidative stress on the
insulin signaling pathway
(102)
I-
Enzymatic antioxidants
· Superoxide dismutase (SOD)
26
Isoforms of SOD are variously located within the cell. Cu/Zn-SOD is
found in both the cytoplasm and the nucleus. Mn-SOD is confined to the
mitochondria, but can be released into the extracellular space
(100)
. SOD
converts superoxide anion radicals produced in the body to hydrogen
peroxide, which is then detoxified to water either by catalase or by
glutathione peroxidase in the lysosomes and mitochondria, respectively
(96)
,
thereby reducing the likelihood of superoxide anion interacting with nitric
oxide to form reactive peroxynitrite
(100)
. However, H
2
O
2
can also be
converted to the highly reactive OH
·
radical in the presence of transition
elements like iron and copper
(103)
.
II-
Non-enzymatic antioxidants
1-
Vitamins
Vitamins A, C and E are diet-derived and detoxify free radicals
directly. They also interact in recycling processes to generate their reduced
forms. -tocopherol is reconstituted, when ascorbic acid recycles the
tocopherol radical generating dihydroascorbic acid, which is recycled by
glutathione
(100)
.
Vitamin E, a fat soluble vitamin, reacts directly with peroxyl and
superoxide radicals to protect membranes from lipid peroxidation
(100)
. It
exists in eight different forms, of which -tocopherol is the most active form
in humans. Hydroxyl radical reacts with tocopherol forming a stabilized
phenolic radical, which is reduced back to the phenol by ascorbate and
NAD(P)H dependent reductase enzymes
(96)
. The deficiency of vitamin E is
concurrent with increased peroxides and aldehydes in many tissues.
However, there have been conflicting reports about vitamin E levels in
diabetic animals and human subjects that its plasma and/or tissue levels are
reported to be unaltered, increased or decreased in diabetes
(100)
.
Vitamin C, ascorbic acid, is an important potent water soluble
antioxidant vitamin in human plasma, acting as an electron donor; it is
capable of scavenging oxygen-derived free radicals and sparing other
endogenous antioxidants from consumption
(104)
. It can increase NO
production in endothelial cells by stabilizing NOS cofactor
tetrahydrobiopterin (BH4)
(96)
. Vitamin C itself is oxidized to
27
dehydroascorbate, which is considered as a marker of oxidative stress, as in
smoking and diabetes mellitus
(105)
. Plasma and tissue levels of vitamin C
are 4050% lower in diabetic compared with non-diabetic subjects
(106)
.
2-
Coenzyme Q
10
(CoQ
10
)
It is an endogenously synthesized lipid soluble antioxidant that acts as
an electron carrier in the complex II of the mitochondrial electron transport
chain and in higher concentrations, it scavenges O
2
·
-
and improves
endothelial dysfunction in diabetes
(83, 96)
.
3-
-Lipoic acid
It is an antioxidant, which can exert beneficial effects in both aqueous
and lipid environments. -lipoic acid is reduced to another active
compound dihydrolipoate, which is able to regenerate other antioxidants,
such as vitamin C, vitamin E and reduced glutathione through redox
cycling
(83, 96)
.
4-
Trace elements
Selenium, an essential trace element, is involved in the complex
defense system against oxidative stress through selenium-dependent
glutathione peroxidases and other selenoproteins
(107)
. It has insulin-
mimetic properties on glucose metabolism both in-vitro and in-vivo, by
stimulating the tyrosine kinases involved in the insulin signaling
cascade
(108)
. Within the context of diabetes mellitus, controversially data on
selenium levels in biological fluids can be found. Lower, similar and even
higher selenium levels were reported in diabetic patients with respect to
healthy subjects
(109)
.
Zinc, magnesium and chromium are of special interest. Severe Zn
deficiency is not frequent but concerns have been raised about Zn levels in
diabetic patients. Some studies have reported Zn deficiency in type 2 diabetes,
others failed to find significant differences with healthy subjects
(110)
. Low
magnesium levels have been associated with increased severity of type 2
diabetes, whereas controversy exists about the importance of
hypomagnesaemia in pre-diabetic states
(110)
. Previous studies also reported that
diabetic patients have a significantly lower plasma chromium levels with higher
28
urinary levels than in healthy subjects. This combination of abnormalities
suggests a chronic renal loss of chromium
(111)
.
Vanadium compounds are one of the most studied substances for the
long-term treatment of diabetes. Vanadium exhibits insulin-mimetic effects
in-vitro and in the streptozotocin diabetic rat with some insulin-enhancing
effects
(112)
.
5-
Glutathione
Glutathione ( -glutamyl-L-cysteinylglycine, GSH), Figure (6), is a
small intracellular ubiquitous tripeptide, which is a sulfhydryl (SH)
antioxidant, antitoxin and enzyme cofactor
(113)
, present in both prokaryotes
and eukaryotes
(114)
. Being water soluble, it is found mainly in the cell
cytosol and other aqueous phases of the living system
(113)
.
Glutathione antioxidant system predominates among other
antioxidants systems due to its very high reduction potential and high
intracellular concentrations compared to other antioxidants in tissues.
Glutathione is found almost exclusively in its thiol-reduced active form
(GSH), comprises 90% of the total low molecular weight thiol in the body
(115)
. GSH often attains millimolar levels inside cells, especially highly
concentrated in the liver and in lens, spleen, kidney, erythrocytes and
leukocytes, however its plasma concentration is in micromolar range
(116)
.
Glutathione is an essential cofactor for antioxidant enzymes, namely
the GSH peroxidases, which serve to detoxify hydrogen peroxide and other
peroxides generated in water phase as well as the cell membranes and other
lipophilic cell phases by reacting them with GSH, which then becomes in
the oxidized form (GSSG). The recycling of GSSG to GSH is
accomplished mainly by the enzyme glutathione reductase using the
coenzyme NADPH as its source of electrons. Therefore NADPH, coming
mainly from the pentose phosphate shunt, is the predominant source of
GSH reducing power
(117)
. Moreover, GSH is an essential component of the
glyoxalase enzyme system, which is responsible for catabolism of the
highly reactive aldehydes; methylglyoxal and glyoxal. It can also bind to
these aldehydes, causing them to be excreted in bile and urine
(115)
. These
effects have particular implications for preventive health, as lipid
29
peroxidation has been found to contribute to the development of many
chronic diseases in humans.
The ratio of reduced to oxidized glutathione (GSH/GSSG) within cells
is often used as a measure of cellular toxicity or vice versa as a predictor of
the antioxidative capacity and redox state of the cells
(114)
. GSH in the body
is synthesized mostly de-novo, with cysteine being the limiting amino acid,
so increasing cysteine supply is necessary to raise GSH synthesis and
concentration. GSH may be a good reservoir for cysteine, as its
concentration in tissues is 5-7 times higher than free cysteine
(118)
.
Figure (6): Structure of GSH ( -glutamylcysteinyl glycine), where the N-
terminal glutamate and cysteine are linked by the -carboxyl
group of glutamate
(119)
.
GSH makes major contributions to the recycling of other antioxidants
that have become oxidized such as -tocopherol, vitamin C and perhaps also
the carotenoids
(117)
. Moreover, GSH is important in the synthesis and repair
of DNA, as it is required in the conversion of ribonucleotides to
deoxyribonucleotides
(120)
.
A major function of GSH is the detoxification of xenobiotics and/or
their metabolites. It is also involved in maintaining the essential thiol status
of many important enzymes and proteins
(117)
. It participates in some
cellular functions as amino acid translocation across the cell membrane
(121)
and folding of newly synthesized proteins
(122)
. In addition, GSH is essential
for the proliferation, growth, differentiation and activation of immune
cells
(117)
and is implicated in the modulation of cell death (cellular
apoptosis and necrosis)
(123)
.
Some oxidative stressors are known for their ability to deplete GSH.
These include smoking, alcohol intake, some over the counter drugs (as
acetaminophen), household chemicals, strenuous aerobic exercise, dietary
30
deficiency of methionine (an essential amino acid and GSH precursor),
ionizing radiation, tissue injury, surgery, trauma, bacterial or viral
infections (as HIV-1) and environmental toxins
(117)
.
GSH reduction has been associated with the pathogenesis of a variety
of diseases; therefore, systemic GSH status could serve as an index of
general health.
Glutathione in liver diseases
GSH depletion is involved in liver injury and enhanced morbidity
related to liver hypofunction. Studies had been demonstrated a decrease in
plasma and liver GSH, increase in GSSG and a significant decrease in
cysteine present in cirrhotic patients, chronic alcoholic and non-alcoholic
liver disease (fatty liver, acute and chronic hepatitis); as compared with the
healthy subjects
(117)
.
Glutathione in immunity and HIV disease
Adequate GSH is essential for mounting successful immune responses
when the host is immunologically challenged. Healthy humans with
relatively low lymphocyte GSH were found to have significantly lower
CD4 counts
(117)
. It was postulated that GSH deficiency could lead to the
progression of immune dysfunction, weight loss, cachexia and wasting
syndrome, which are known AIDS stigmas. GSH depletion is also seen in
many autoimmune diseases as Crohn's disease, an inflammatory
immunomediated disorder, in which low GSH, elevated GSSG levels and
altered GSH enzymes were found in the affected ileal zones
(114)
.
Glutathione in diabetes mellitus
Low blood thiol status and reduced systemic GSH content were
reported in diabetic and glucose intolerant patients as a result of insulin
deficiency. It was reported that chronic hyperglycemia resulted in enhanced
apoptosis in human endothelial cells, which was attenuated by insulin due
to its ability to induce glutamate cysteine ligase expression
(119)
. Platelets
from diabetics have lower GSH levels and make excess thromboxane
(TxA2), thus having a lowered threshold for aggregation. This may
Details
- Pages
- Type of Edition
- Erstausgabe
- Year
- 2015
- ISBN (eBook)
- 9783954898534
- ISBN (Softcover)
- 9783954893539
- File size
- 6.7 MB
- Language
- English
- Publication date
- 2015 (January)
- Keywords
- type evaluation l-cysteine
