Free beta cells of the pancreas. As a

Free radicals are molecules that
have one or more unpaired electrons meaning they have to gain or donate an
electron to secure stability. They have the capability to form injurious bonds
with proteins, lipids and carbohydrates, thus causing cellular damage when they
are in high concentrations. (Ha et al,
1999) Free radicals that are produced from the activation of oxygen are known
as reactive oxygen species (ROS). There are two ways activation of oxygen can
occur: 1. Absorption of enough energy to cause one of the unpaired electrons to
spin in the reverse direction resulting in it becoming a singlet oxygen in
which the electrons have opposite spins. 2. Stepwise monovalent reduction of
oxygen to form superoxide, H2O2, hydroxyl radical then H2O which can be seen in
Figure 1. (Matough et al, 2012) ROS
can be charged in the case of the hydroxyl radical, or uncharged with the
example of hydrogen peroxide. The balance of free radical production and
elimination is important for homeostasis. If the rate of generation exceeds the
rate of elimination, oxidative stress occurs which is when the body’s ability
to counteract or detoxify the free radicals using antioxidants is deemed ineffective.
(Matough et al, 2012)




Figure 1. Stepwise activation of Oxygen. (Mozzaffarieh et al, 2008)

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Diabetes Mellitus Type 1 (DMT1)
is an autoimmune disorder that sees the destruction of the insulin-producing
beta cells of the pancreas. As a result of low insulin production, the affected
individuals are unable to remove glucose from the blood leaving them in a state
of hyperglycaemia. In hyperglycaemic conditions, various types of vascular
cells are able to form ROS leading to a state of oxidative stress. (Ha et al, 1999) Studies, with both animals
and diabetic patients, have shown that elevated extracellular and intracellular
glucose concentrations result in oxidative stress. (West IC, 2000) ROS deplete
antioxidant defences leaving the tissues more susceptible to oxidative damage
by reacting with lipids in cellular membranes, nucleotides in DNA, sulfhydryl
groups in proteins and cross-linking fragmentations of ribonucleoproteins
leading to changes in cellular structure and function. (Ashan et al, 2003) The many damaging abilities
of ROS that lead to oxidative stress are what suggests they play a key role in
the pathogenesis of Diabetes Mellitus Type 1 (DBT1) associated complications
which is what is to be discussed throughout this essay. (Rosen et al, 2001)

In DMT1 there are a number of
mechanisms that can cause oxidative stress either by altering the redox balance
or direct generation of free radicals. This is thought to occur via mechanisms
including increased polyol pathway flux, increased intracellular formation of
advanced glycation end-products, activation of protein kinase C, or
overproduction of superoxide by the mitochondrial electron transport chain. (Ahmad
et al, 2005)

The polyol pathway leads to
reduction of glucose via aldose reductase to produce sorbitol in a NADPH
dependent manner. Sorbitol is oxidised to fructose by the enzyme sorbitol
dehydrogenase, with NAD+ reduced to NADH. The major function of aldose
reductase is to reduce toxic aldehydes formed by ROS into inactive alcohols. In
normal conditions, very little glucose is transformed into sorbitol as aldose
reductase has a low affinity for glucose. Under hyperglycaemic conditions seen
in DMT1, enzyme activity is increased therefore, so is the amount of sorbitol
produced. As a consequence, NADPH concentration is decreased which is an
essential co-factor for the production of GSH (a critical intracellular
antioxidant). (Brownlee, 2001). Reduced availability of antioxidants means that
the host is less effective against hyperglycaemic-induced ROS.

Another key mechanism is the excess
free radical production from a variety of sources including the autoxidation of
glucose molecules. (Matough et al, 2012)
In healthy cells ROS levels are under tight regulation by antioxidant enzymes
and non-enzymatic antioxidants. DMT1 leads to excessive cellular levels of ROS
production due to the individual being in a state of hyperglycaemia. (Matough et al, 2012)

An alternative mechanism of ROS
production is the glycation of proteins and antioxidative enzymes leading to an
increased production of advanced glycated end products (AGEs).  (Ahmad et
al, 2005) Covalent binding of an aldehyde or ketone of a sugar to a free
amino group of a protein creates an intermediate that spontaneously rearranges
itself. These are directly converted into AGEs which have the ability to signal
through a cell via a surface receptor ‘RAGE’. (Rains & Jain, 2010) A major
consequence of a ligand bound to the RAGE is the production of intracellular
ROS via the activation of a NADPH oxidase system. The ROS produced then in turn
activates the MAPK pathway which ultimately activates NF-kB. (Brownlee, 2001)
Activation of NF-kB results in the expression of many gene products that are closely
linked with DMT1 associated complications. Furthermore, the glycation of
antioxidative enzymes result in a reduced capacity to detoxify ROS giving rise
to further free radical production. (Matough et al, 2012)

In diabetic individuals, ROS can
also be generated by the activation of the DAG-PKC pathway. (Ahmad et al, 2005) The protein kinase C (PKC)
family consist of a number of different isoforms which are mainly activated by
the messenger DAG. Under hyperglycaemic conditions a cascade of reactions occur
leading to the increased synthesis of DAG, hence increased activation of the
PKC isoforms which can result in a number of alterations in cell signalling. The
result of the alterations in cell signalling can lead to direct production of
ROS or the indirect production of ROS by activating other pathways. (Rains
& Jain, 2010) PKC contributes to matrix protein accumulation by inducing
expression of TGF-B1, fibronectin and collagen which is thought to be a cause
of diabetic nephrology.

A prominent mechanism for ROS
production in DMT1 sufferers is the overproduction of superoxide by the
mitochondrial electron transport chain (ETC). (Brownlee, 2001) In normal glucose
metabolism, the glucose is transformed via glycolysis initiating a series of
reactions which terminates at the ETC. Electrons are transferred through the
ETC and the energy is used to move protons across the membrane. This creates a
voltage across the inner and outer membrane of the mitochondria which is used
for the ATP synthesis. Under hyperglycaemic conditions seen in diabetics, the
number of substrates entering the cycle is increased so consequently number of
reducing equivalents increase too. Once the ETC reaches a threshold voltage,
the electrons are backed up before they begin being donated to the molecular
oxygen hence, superoxide production is increased. (Brownlee, 2001)

Cells in the body contain
antioxidant defence mechanisms which are used for homeostasis of ROS. Diabetes
is associated with reduced levels of antioxidants such as GSH, vitamin C and E.
(Jain, 1998) Glycation of these antioxidative enzymes in diabetes can impair
cellular defence mechanisms resulting in the development of oxidative stress
which contributes to the complications associated with DMT1. Protein glycation
not only affects the antioxidant system but also normal functions of proteins
resulting in alter cell functions in diabetes.

These mechanisms act in a way to
either increase ROS production or decrease the effectiveness of the role of
antioxidants leading to the imbalance necessary for oxidative stress to occur.
Some of the complications associated with oxidative stress in DMT1 include
neuropathy, nephropathy, retinopathy and accelerated coronary heart disease.  

An important role of oxidative
stress in the development of nephrology and neurological complications has been
suggested by studies that have established a causal relationship between the
two. (Sharma et al, 2007) Lipid
peroxides are an indices of oxidative tissue damage, which increase in number
in the kidneys of diabetic mice indicating that the damage is caused in a
ROS-dependent manner. NADPH is a key source of ROS production in diabetes.
Oxidase is located in the plasma membrane of various renal cells including
mesangial. NADPH oxidase-dependent overproduction of ROS has an important role
in promoting hyperglycaemia-induced oxidative stress. (Matough et al, 2012) Furthermore, oxidative
stress causes mRNA expression of TGF-b1 and fibronectin which are genes that
are highlighted in glomerular activity of diabetic individuals. Finally, the inhibition
of oxidative stress relief molecules, such as antioxidants, is the final
mechanism that established in the clinical studies that is associated with
diabetic nephropathy. As all of these conclusions are linked with diabetic
nephropathy and are demonstrated in clinical studies, conclusions are able to
be drawn that oxidative stress has a causal relationship with nephropathy as a
complication of DMT1. (Ha et al, 1999)

Multiple studies suggest that ROS
can react with amino acid residues in vitro generating proteins that may be
denatured, modified or non-functioning. (Turko, 2001) Diabetic hyperglycaemia
causes protein glycation and oxidative degeneration. The degree of which the
protein is glycated is assessed by the evaluation of biomarkers such as
glycated haemoglobin levels. (Ullah et
al, 2016) Glycation of proteins interferes with the proteins’ normal
functions by disrupting molecular conformation, altering enzymatic activity and
interfering with the receptor functioning. (Singh et al, 2014) The non-enzymatic modification of the plasma proteins
such as albumin produce various negative effects including alteration in drug
binding in the plasma, platelet activation, free radical generation, and more.
See figure 2.  (Singh et al, 2014) In addition, glycation of
proteins such as albumin and collagen have had it suggested that they
contribute to vascular stiffness by alteration of the vascular structure and
function. (Goh & Cooper, 2008)



Figure 2. The effects of long term exposure to elevated glucose exposure causing
glycation of proteins.

(Singh et al, 2014)






Lipid hydroperoxides (LHP) are generated
via intermediate reactions of long-chain polyunsaturated fatty acid precursors
that involve oxygen and metal cations. The result of these reactions is the
production of highly reactive lipid radicals which generate further LHPs due to
their close proximity to other lipids in the phospholipid membranes. (Nishigaki
et al, 1981) In addition, diabetes
causes disturbances of lipid profiles, especially by making them more
susceptible to lipid peroxidation. (Lyons, 1991) It is suggested from
experimental studies that polyunsaturated fatty acids (PUFA) are particularly
susceptible to attack by free radicals. This is due to the multiple double bonds
they contain. (Esterbauer et al, 1991)
Hydroxyl radicals remove a hydrogen atom from one of the carbon atoms from the
PUFA and lipoproteins which sees the initiation of a free radical chain
reaction. Thus, causing lipid peroxidation characterized by membrane protein
damage. (Halliwell, 1995) Several studies conclude that diabetic patients had
increased LDL oxidation in comparison to their corresponding controls. Oxidised
lipids can affect cell function as their accumulation in the cell membrane can
result in the leakage of the plasma lemma and interference with the function of
membrane-bound receptors. (Cai et al,
2000) Furthermore, the by-products of lipid peroxidation have cytotoxic and
mutagenic properties.

Oxidative stress is known to
cause oxidation of DNA. It leads to the conversion of deoxyguanosine to 8-oxo,
2-deoxyguanosine in DNA. (Pan et al, 2000)
Concentrations of the latter are used to assess the amount of oxidative stress
in a cell. Studies have shown that DNA damage is significantly higher in
diabetic patients than their healthy corresponding controls, and significantly
higher again in patients with diabetic nephropathy. (Pan et al, 2000) DNA damage has been linked to certain diseases such as
cancer, meaning DMT1-induced DNA damage can leave an individual more
susceptible to other damaging diseases. Moreover, DNA damage can also lead to
the production of non-functional proteins and errors in the replication of
future cell lines.

To conclude, there is
considerable research and evidence to support that induction of oxidative
stress due to ROS formation is a key process in the onset of diabetic
complications. The exact mechanisms of ROS-induced oxidative stress in the
induction of diabetic complications is only partially known, thus more research
needs to be invested into gaining a full understanding of the pathogenesis of
these DMT1 associated conditions. On the contrary, there are many ethical
issues surrounding research into diabetes due to necessity of completing
potentially harmful procedures out on animal subjects.