Free radicals are molecules thathave one or more unpaired electrons meaning they have to gain or donate anelectron to secure stability. They have the capability to form injurious bondswith proteins, lipids and carbohydrates, thus causing cellular damage when theyare in high concentrations. (Ha et al,1999) Free radicals that are produced from the activation of oxygen are knownas reactive oxygen species (ROS).
There are two ways activation of oxygen canoccur: 1. Absorption of enough energy to cause one of the unpaired electrons tospin in the reverse direction resulting in it becoming a singlet oxygen inwhich the electrons have opposite spins. 2. Stepwise monovalent reduction ofoxygen to form superoxide, H2O2, hydroxyl radical then H2O which can be seen inFigure 1.
(Matough et al, 2012) ROScan be charged in the case of the hydroxyl radical, or uncharged with theexample of hydrogen peroxide. The balance of free radical production andelimination is important for homeostasis. If the rate of generation exceeds therate of elimination, oxidative stress occurs which is when the body’s abilityto 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) Diabetes Mellitus Type 1 (DMT1)is an autoimmune disorder that sees the destruction of the insulin-producingbeta cells of the pancreas. As a result of low insulin production, the affectedindividuals are unable to remove glucose from the blood leaving them in a stateof hyperglycaemia. In hyperglycaemic conditions, various types of vascularcells are able to form ROS leading to a state of oxidative stress.
(Ha et al, 1999) Studies, with both animalsand diabetic patients, have shown that elevated extracellular and intracellularglucose concentrations result in oxidative stress. (West IC, 2000) ROS depleteantioxidant defences leaving the tissues more susceptible to oxidative damageby reacting with lipids in cellular membranes, nucleotides in DNA, sulfhydrylgroups in proteins and cross-linking fragmentations of ribonucleoproteinsleading to changes in cellular structure and function. (Ashan et al, 2003) The many damaging abilitiesof ROS that lead to oxidative stress are what suggests they play a key role inthe pathogenesis of Diabetes Mellitus Type 1 (DBT1) associated complicationswhich is what is to be discussed throughout this essay. (Rosen et al, 2001)In DMT1 there are a number ofmechanisms that can cause oxidative stress either by altering the redox balanceor direct generation of free radicals. This is thought to occur via mechanismsincluding increased polyol pathway flux, increased intracellular formation ofadvanced glycation end-products, activation of protein kinase C, oroverproduction of superoxide by the mitochondrial electron transport chain.
(Ahmadet al, 2005)The polyol pathway leads toreduction of glucose via aldose reductase to produce sorbitol in a NADPHdependent manner. Sorbitol is oxidised to fructose by the enzyme sorbitoldehydrogenase, with NAD+ reduced to NADH. The major function of aldosereductase is to reduce toxic aldehydes formed by ROS into inactive alcohols. Innormal conditions, very little glucose is transformed into sorbitol as aldosereductase has a low affinity for glucose.
Under hyperglycaemic conditions seenin DMT1, enzyme activity is increased therefore, so is the amount of sorbitolproduced. As a consequence, NADPH concentration is decreased which is anessential co-factor for the production of GSH (a critical intracellularantioxidant). (Brownlee, 2001). Reduced availability of antioxidants means thatthe host is less effective against hyperglycaemic-induced ROS.Another key mechanism is the excessfree radical production from a variety of sources including the autoxidation ofglucose molecules. (Matough et al, 2012)In healthy cells ROS levels are under tight regulation by antioxidant enzymesand non-enzymatic antioxidants.
DMT1 leads to excessive cellular levels of ROSproduction due to the individual being in a state of hyperglycaemia. (Matough et al, 2012) An alternative mechanism of ROSproduction is the glycation of proteins and antioxidative enzymes leading to anincreased production of advanced glycated end products (AGEs). (Ahmad etal, 2005) Covalent binding of an aldehyde or ketone of a sugar to a freeamino group of a protein creates an intermediate that spontaneously rearrangesitself. These are directly converted into AGEs which have the ability to signalthrough a cell via a surface receptor ‘RAGE’. (Rains & Jain, 2010) A majorconsequence of a ligand bound to the RAGE is the production of intracellularROS via the activation of a NADPH oxidase system. The ROS produced then in turnactivates the MAPK pathway which ultimately activates NF-kB. (Brownlee, 2001)Activation of NF-kB results in the expression of many gene products that are closelylinked with DMT1 associated complications.
Furthermore, the glycation ofantioxidative enzymes result in a reduced capacity to detoxify ROS giving riseto further free radical production. (Matough et al, 2012) In diabetic individuals, ROS canalso 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 bythe messenger DAG. Under hyperglycaemic conditions a cascade of reactions occurleading to the increased synthesis of DAG, hence increased activation of thePKC isoforms which can result in a number of alterations in cell signalling. Theresult of the alterations in cell signalling can lead to direct production ofROS or the indirect production of ROS by activating other pathways. (Rains& Jain, 2010) PKC contributes to matrix protein accumulation by inducingexpression of TGF-B1, fibronectin and collagen which is thought to be a causeof diabetic nephrology. A prominent mechanism for ROSproduction in DMT1 sufferers is the overproduction of superoxide by themitochondrial electron transport chain (ETC).
(Brownlee, 2001) In normal glucosemetabolism, the glucose is transformed via glycolysis initiating a series ofreactions which terminates at the ETC. Electrons are transferred through theETC and the energy is used to move protons across the membrane. This creates avoltage across the inner and outer membrane of the mitochondria which is usedfor the ATP synthesis. Under hyperglycaemic conditions seen in diabetics, thenumber of substrates entering the cycle is increased so consequently number ofreducing equivalents increase too.
Once the ETC reaches a threshold voltage,the electrons are backed up before they begin being donated to the molecularoxygen hence, superoxide production is increased. (Brownlee, 2001)Cells in the body containantioxidant defence mechanisms which are used for homeostasis of ROS. Diabetesis associated with reduced levels of antioxidants such as GSH, vitamin C and E.(Jain, 1998) Glycation of these antioxidative enzymes in diabetes can impaircellular defence mechanisms resulting in the development of oxidative stresswhich contributes to the complications associated with DMT1. Protein glycationnot only affects the antioxidant system but also normal functions of proteinsresulting in alter cell functions in diabetes.
These mechanisms act in a way toeither increase ROS production or decrease the effectiveness of the role ofantioxidants leading to the imbalance necessary for oxidative stress to occur.Some of the complications associated with oxidative stress in DMT1 includeneuropathy, nephropathy, retinopathy and accelerated coronary heart disease. An important role of oxidativestress in the development of nephrology and neurological complications has beensuggested by studies that have established a causal relationship between thetwo. (Sharma et al, 2007) Lipidperoxides are an indices of oxidative tissue damage, which increase in numberin the kidneys of diabetic mice indicating that the damage is caused in aROS-dependent manner. NADPH is a key source of ROS production in diabetes.Oxidase is located in the plasma membrane of various renal cells includingmesangial.
NADPH oxidase-dependent overproduction of ROS has an important rolein promoting hyperglycaemia-induced oxidative stress. (Matough et al, 2012) Furthermore, oxidativestress causes mRNA expression of TGF-b1 and fibronectin which are genes thatare highlighted in glomerular activity of diabetic individuals. Finally, the inhibitionof oxidative stress relief molecules, such as antioxidants, is the finalmechanism that established in the clinical studies that is associated withdiabetic nephropathy. As all of these conclusions are linked with diabeticnephropathy and are demonstrated in clinical studies, conclusions are able tobe drawn that oxidative stress has a causal relationship with nephropathy as acomplication of DMT1. (Ha et al, 1999)Multiple studies suggest that ROScan react with amino acid residues in vitro generating proteins that may bedenatured, modified or non-functioning.
(Turko, 2001) Diabetic hyperglycaemiacauses protein glycation and oxidative degeneration. The degree of which theprotein is glycated is assessed by the evaluation of biomarkers such asglycated haemoglobin levels. (Ullah etal, 2016) Glycation of proteins interferes with the proteins’ normalfunctions by disrupting molecular conformation, altering enzymatic activity andinterfering with the receptor functioning. (Singh et al, 2014) The non-enzymatic modification of the plasma proteinssuch as albumin produce various negative effects including alteration in drugbinding in the plasma, platelet activation, free radical generation, and more.See figure 2. (Singh et al, 2014) In addition, glycation ofproteins such as albumin and collagen have had it suggested that theycontribute to vascular stiffness by alteration of the vascular structure andfunction. (Goh & Cooper, 2008) Figure 2.
The effects of long term exposure to elevated glucose exposure causingglycation of proteins. (Singh et al, 2014) Lipid hydroperoxides (LHP) are generatedvia intermediate reactions of long-chain polyunsaturated fatty acid precursorsthat involve oxygen and metal cations. The result of these reactions is theproduction of highly reactive lipid radicals which generate further LHPs due totheir close proximity to other lipids in the phospholipid membranes.
(Nishigakiet al, 1981) In addition, diabetescauses disturbances of lipid profiles, especially by making them moresusceptible to lipid peroxidation. (Lyons, 1991) It is suggested fromexperimental studies that polyunsaturated fatty acids (PUFA) are particularlysusceptible to attack by free radicals. This is due to the multiple double bondsthey contain. (Esterbauer et al, 1991)Hydroxyl radicals remove a hydrogen atom from one of the carbon atoms from thePUFA and lipoproteins which sees the initiation of a free radical chainreaction. Thus, causing lipid peroxidation characterized by membrane proteindamage. (Halliwell, 1995) Several studies conclude that diabetic patients hadincreased LDL oxidation in comparison to their corresponding controls. Oxidisedlipids can affect cell function as their accumulation in the cell membrane canresult in the leakage of the plasma lemma and interference with the function ofmembrane-bound receptors. (Cai et al,2000) Furthermore, the by-products of lipid peroxidation have cytotoxic andmutagenic properties.
Oxidative stress is known tocause 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 stressin a cell. Studies have shown that DNA damage is significantly higher indiabetic patients than their healthy corresponding controls, and significantlyhigher again in patients with diabetic nephropathy.
(Pan et al, 2000) DNA damage has been linked to certain diseases such ascancer, meaning DMT1-induced DNA damage can leave an individual moresusceptible to other damaging diseases. Moreover, DNA damage can also lead tothe production of non-functional proteins and errors in the replication offuture cell lines. To conclude, there isconsiderable research and evidence to support that induction of oxidativestress due to ROS formation is a key process in the onset of diabeticcomplications. The exact mechanisms of ROS-induced oxidative stress in theinduction of diabetic complications is only partially known, thus more researchneeds to be invested into gaining a full understanding of the pathogenesis ofthese DMT1 associated conditions. On the contrary, there are many ethicalissues surrounding research into diabetes due to necessity of completingpotentially harmful procedures out on animal subjects.