There are many complications of DM due to physiological damage caused by over-stimulation of alternative pathways to manage persistent hyperglycaemia, however diabetic neuropathy (DN) is the most common of the complications[1][6]. DN affects just over 50% of both IDDM and NIDDM sufferers with almost equal prevalence in both types, being slightly more common in NIDDM[6][7]. DN is most commonly seen in patients that have had diabetes for over 25 years and those that do not have good glycaemic control[1]. DN is characterised by damage to sensory and motor nerve fibres[6] but mostly affects the sensory neurones resulting in a loss of feeling[1][7], the condition is usually chronic and progressive[6]. It tends to primarily present in the peripheral neurones in the feet and later in the hands[1][6]. This inability to feel pain leaves the patient less able to monitor any progressive trauma to the foot and therefore results in ulcers in a condition called 'diabetic foot'[1][6] and is therefore the leading cause of non-traumatic limb amputations[6].
Fig.1. Image taken from [6] and shows areas affected by peripheral diabetic neuropathy.
DN is caused by persistent hyperglycaemia and initiation of some key pathways that disturb the metabolic and oxidative stress levels in cells leading to damage[6]. Decreased blood supply to the nerves caused by the osmotic affects of hyperglycaemia also amplifies these affects[6]. The key pathways involved are the polyol pathway, advanced glycation end-product (AGE) formation and protein kinase C (PKC) activation and also increased use of the hexosamine pathway[4]. Although these biochemical pathways are deleterious in their own right, together they produce excess reactive oxygen species (ROS) by imbalancing the oxidation and reduction states of mitochondria and increasing oxidative stress within the cell[6]. This oxidative stress increases the production of molecules inducing inflammation and neuronal dysfunction[6].
The Polyol Pathway
The polyol pathway is one of the keys to understanding the development of cellular damage in hyperglycaemia as it produces some of the major molecules that cause oxidative stress in diabetes[8]. Under normal conditions the polyol pathway is used to convert aldehydes to inactive alcohols and works as a protective measure for cells[4], however the pathway is initiated in hyperglycaemia[8].
Fig.2. Shows the polyol pathway under normal conditions. Image derived from Medbio Info, 'Blood sugar in health and disease.' www.medbio.info/Horn/Time%205/sugar.htm . Accessed 16.02.2010.
When glucose enters the pathway it is reduced to sorbitol by the enzyme aldose reductase by using the co-factor NADPH[8]. This is then further reduced to fructose by the enzyme sorbitol dehydrogenase also using NADPH as its co-factor[4][8]. Aldose reductase does not usually have a high affinity for glucose under normoglycaemic conditions but when excess glucose in hyperglycaemia is metabolised in the pathway it leads to intracellular sorbitol accumulation and a depletion of NADPH[4]. NADPH is needed to restore intracellular glutathione which is a natural antioxidant found in the body and has been found to reduce lipid peroxidation of cell membranes[9]. This inability to regenerate glutathione and neutralise reactive oxygen species (ROS) results in oxidative stress[9] and contributes towards cellular loss and damage in diabetic neuropathy[8].
Excessive products of the polyol pathway due its over-stimulation caused by hyperglycaemia negatively affect the oxidative stress levels of the cell[8]. Sorbitol accumulation in cells has an osmotic effect as it is a hydrophilic alcohol and therefore does not cross cell membranes easily[8]. Fructose produced as a result of the polyol pathway can be phosphorylated to make fructose-3-phosphate, both these molecules are potent stimulants in the formation of advanced glycation end-products (AGEs)[8] and also initiating the PKC pathway by forming diaceylglycerol (DAG)[6]. The polyol pathway is mostly associated with being the major contributing factor towards diabetic retinopathy but is also a major pathway in the pathogenesis of neuropathy[8].
Activation of the PKC pathway due to increased DAG production in the polyol pathway is also thought to contribute to pathophysiology of nerve cells in diabetic neuropathy[6]. Increase in PKC pathway activity affects many cell types but in particular alters the Na+/K+ ATPase pumps and intracellular enzymes in smooth muscle cells and neurones and therefore interferes with nerve conduction[6][10]. This interference results in the loss of feeling characteristic of diabetic peripheral neuropathy.
Advanced Glycation End-products (AGEs)
AGEs are modified intracellular and extracellular biomolecules and are thought to be the pivotal molecules for leading to oxidative stress in diabetic disease states[6]. As a result of intracellular hyperglycaemia, pre-cursors to AGEs, dicarbonyls, are formed during the oxidation of glucose to glyoxal[4][6]. These dicarbonyls react with the amine groups on intracellular proteins and form AGEs[4]. Methylglyoxal (MG) is a highly potent dicarbonyl and increases sensitivity of cells to damage[6]. AGEs may modify functions of proteins they bind to by causing them to cross-link extracellular matrix molecules or binding them to AGE receptors (RAGE) on other cells such as macrophages, endothelial cells and smooth muscle cells[6]. Those cells with RAGE are particularly associated with atherosclerosis[6] and so elevated AGE levels have been linked with the increased incidence of cardiovascular disease in diabetic patients[11].
AGEs interact with RAGE and causes activation of mitogen-activated protein kinases (MAPK), protein kinase C and nuclear factor kappa B (NF-ï«B). NF-ï«B translocates to the nucleus where its p65 subunit[12] up-regulates transcription of adhesion molecules and pro-inflammatory molecules such as ICAM, IL-6 and TNF[4][9][12]. NF-ï«B formed as a result of increased AGEs in hyperglycaemia is related to diabetic neuropathy in that peripheral nerve myelin is a target for macrophages activated and modified by RAGE[13]. Damage occurs by macrophages phagocytosising the nerve myelin and AGEs modifying the intracellular matrix proteins; tubulin, neurofilament and actin, by cross-linking them[9][13]. These mechanisms along with the glycation of the extracellular matrix protein laminin, leads to demyelination and impaired nerve function and blood flow, resulting in loss of feeling in the periphery nerve a condition known as diabetic neuropathy[9][11][12][14].
Oxidative Stress
Oxidative stress involves a wide interaction of the various pathways and mechanisms already mentioned and there is much evidence to indicate that hyperglycaemia is the initiator that underpins all these mechanisms. Oxidative stress indicates the creation of free radicals and reactive oxygen species (ROS) these are usually damaging to human tissues, however ROS are produced as a part of normal everyday metabolism[15]. Is it known that hyperglycaemia increases ROS production in mitochondria[15], usually NADH and FADH2 produced as a result of the tricarboxylic acid cycle move to the mitochondria and donate electrons to the mitochondria redox enzyme complexes in the membrane[6]. Donated electrons are then transferred through the membrane complex in the mitochondria and join oxygen to form water as a by-product[6]. This electron transporting system creates a proton gradient between the inner and outer mitochondrial membranes, the gradient of which is crucial for ATP synthesis and consequently mitochondrial survival[6]. ROS is formed as a natural by-product of the electrons transferring from subunit II to subunit III of the mitochondrial membrane complex but is usually produced at low levels and is metabolised by the antioxidant glutathione to rid it of its deleterious effects[6].
Fig. 3. Image taken from [6]. Image shows the production of ROS by the mitochondria-membrane complex and its damage on the mitochondria DNA and membrane.
However, during hyperglycaemia the mitochondria as a consequence metabolises more glucose through the glycolytic and tricarboxylic acid cycles, producing more NADH and FADH[6][16]. This disturbs the delicate proton gradient needed to produce ATP and causes a high proton gradient on the inner mitochondria membrane[6]. This increases the mitochondrial membrane complex activity and increases the amount of ROS produced as a by-product, resulting in oxidative stress[6][16].
ROSs deleterious effects include damage to the mitochondria and cell DNA and membrane, but these effects are usually counterbalanced by antioxidants such as glutathione in the body by donating an electron[6]. As already described above, the imbalance of antioxidant ability and ROS production brings about oxidative stress[16] and it's the depletion of intracellular NADPH caused by the accumulation of sorbitol from the polyol pathway that diminishes the ability of glutathione to neutralise ROS by donating an electron[6]. Increased intracellular ROS levels caused by both the polyol pathway activity and AGE formation enhances mitochondria damage and subsequently axon destruction and apoptosis[6]. Neurones are particularly prone to damage as they have a high quantity of mitochondria and a direct blood supply[6].
Methylglyoxal
Methylglyoxal is a significant AGE precursor therefore being a popular candidate for research into oxidative stress in diabetic neuropathy[9][17]. Methylglyoxal (MG) is formed as a result of high intracellular levels of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate from hyperglycaemia[17]. MG has been found in high concentrations in diabetic patients and this also corresponds with patients with diabetic complications[18]. MG is a highly reactive dicarbonyl and is a target for research in oxidative stress in diabetes as it thought to have a key role in the cross-linking of intracellular matrix proteins[9][13][19]. Usually MG is metabolised by the glyoxalase system to prevent its deleterious effects, enzymes used in this process are glyoxalase I and II both using glutathione as a co-factor[19]. Therefore when glutathione activity is reduced due to sorbitol accumulation and NADPH depletion, the detrimental effects of MG can't be neutralised[4].
As MG is an AGE it modifies proteins and alters their function, one particular consequence of this is the modification of the inhibitory molecule Sp3 which suppresses the molecule angiotensin II[6]. This as a result activates endothelial cells including in the endoneurium which is a thin layer of endothelial cells enveloping each peripheral nerve fibre[20]. The activation of the endoneurium results in increased production of ROS due to the growing number of macrophages attracted to the area and their production of cytokines and reduced blood flow to the area[6]. Increased ROS concentrations in the endothelium surrounding the neurones lead to oxidative stress, demyelination and neuronal apoptosis[6].
Neuronal apoptosis caused by ROS and oxidative stress specifically describes the death of Schwann cells surrounding the neurone[21]. Schwann cells surround peripheral neurones and provide insulation which is important for conduction of electrical impulses and are crucial for neuronal survival[22]. The condition of diabetic neuropathy encompasses the damage and death of Schwann cells leading to the loss of feeling in peripheral areas[21]. The mechanism in the process of Schwann cell apoptosis has been found to be that MG produced in hyperglycaemia activates the p38 MAPK and JNK pathway and causes apoptosis of the Schwann cell[21].
Antioxidants
Knowing the mechanisms involved in the pathogenesis of diabetic neuropathy, the leading therapeutic approach is to alleviate oxidative stress and given the nature of antioxidants, research in to their effects on oxidative stress is a plausible route. A large proportion of the body's antioxidant supply used for defence against oxidative stress is taken in through the diet in the form of micronutrients[6]. The mechanism of defence from antioxidants is due to the antioxidant enzymes they produce to remove ROS molecules and prevent oxidation of cells[6]. Glutathione is a natural antioxidant found in the body[9] and as well as sorbitol indirectly depleting it's antioxidant activity, MG directly reduces its effects by covalently binding to it[23]. Studies in to the prevention of apoptosis on methylglyoxal-induced Neuro-2A cells using antioxidants such as phenolics acids showed that they were successful in reducing the affects of methylglyoxal[23].
Phenolic acids are found in a wide range of higher plants, higher plants being those that have their reproductive organs in flowers and fruit[24]. A major group of phenolic acids are the flavonoids, one of the antioxidants included in the flavonoid group is vanillic acid[23]. Vanillic acid provides the colour and flavour for many fruits and vegetables as well as beverages such as tea and wine[25]. It is thought that because of the antioxidant qualities of phenolic acids reducing lipid peroxidation and ROS levels they help to prevent disease states such as cardiovascular disease, hypertension, cancer and oxidative stress[25][26].
A phenolic acid is a compound that in its chemical make-up has an aromatic ring with one or more hydroxyl replacements, they also attach to sugars making them hydrophilic[25]. Flavonoids are composed of two phenolic rings joined by a three carbon unit[25]. They are a number of ways that free radicals cause cellular damage, the main mechanism is by carbon and oxygen radicals combining and forming peroxyl radicals that attack fatty acids in the cell lipid membrane[25]. This prompts a peroxidation chain of reactions of lipids where flavonoid can interrupt and stop the chain reaction[25]. Therefore for a molecule to be classed as an antioxidant it must be able to stop or impede the oxidation of a compound and make the resulting radical stable[25].
Phenolic acids have been suggested as being one of the most influential antioxidants in alleviating oxidative stress and preventing diabetic neuropathy due to its quality of free-radical scavenging[23]. A study in to the protective effects of phenolic acids was undertaken on methylglyoxal-induced apoptosis of Neuro-2A cells[23]. The antioxidants used were syringic acid, chlorogenic acid and vanillic acid as in preliminary experiments they were found to be the most inhibitory of the p38 MAPK pathway that leads to apoptosis of the Schwann cells[23]. The cell lines used were Neuro-2A cells which are neuroblastoma cells, they were exposed to 400ïM MG and 10, 20 and 50ïM of the three different phenolic acids[23]. Results showed that all the antioxidants increased cell viability and decreased apoptosis of cells, among other effects when exposed to methylglyoxal[23].
Fig. 2. Image taken from the results from Huang et al in the Cytoprotective effects of phenolic acids on methylglyoxal-induced apoptosis in Neuro-2A cells[23]. The graph shows a significant decrease in the amount of apoptotic cells compared to the methylglyoxal-exposed cells without antioxidants.
Results also showed that phosphorylation levels of the proteins p38 and JNK were induced when cells were exposed to methylglyoxal, this signifies that the p38 MAPK apoptosis pathway had increased activity, relating to the increased percentage of apoptotic cells in the sample(Fig. 2.)[23]. This experiment, among others have shown that phenolic acids have reduced the effects of methylglyoxal on the apoptosis rates of Schwann cells in diabetic neuropathy[23][27].
Current research
Further investigations in to the biological effects of vanillic acid were carried out by the same scientific team to look more in-depth at how vanillic acid works[28]. Out of the fourteen phenolic acids studied they found that vanillic acid had the best cytoprotective abilities overall[28]. In their study they found that vanillic acid significantly reduced ROS production in cells exposed to 400ïM methylglyoxal and ranging concentrations of vanillic acid from 20, 50 and 100ïM for 72 hours[28]. They also found that vanillic acid reduced the formation of the methylglyoxal-induced AGE carboxymethyl lysine (CML) and therefore blocks intracellular methylglyoxal-stimulated glycation, increasing ROS[28]. Results yielded from these studies show promising advances in to the treatment of diabetic neuropathy, however is the use of Neuro-2A cells a good enough indicator of the long-term pathogenesis of mature neurones? Studies have shown that may be Neuro-2A cells do not fully represent adult neurones as they lack maturity, other cell lines may have better representation[29].
In vitro vs in vivo
There have been many studies representing in vitro and in vivo effects of diabetic neuropathy including the use of Streptozotocin to induce diabetes in a range of small rodents including rats, mice and Chinese hamsters to study the in vivo effects of diabetic neuropathy because they bear some similarity to human mechanisms[29]. However in in vivo experiments it is difficult to measure short-term signal pathways during pathogenesis and tend to look more at resulting cellular and systematic effects of diabetes[29]. In vitro experiments like the ones adopted by Huang et al[23][28] can go some way to bridging the gap of knowledge around intracellular pathways seen in diabetic neuropathy that can't be studied extensively in in vivo experiments[29].
Primary cultured cells taken directly from diabetic-induced rodents, although representing human disease mechanisms, have been criticised due to the length of time taken to obtain a sample, time which may include vital events of cellular metabolism[29]. To overcome these problems dorsal root ganglions from mammalian embryos have been primary cultured and are seen to represent in vivo activity as well as being able to show detailed time scales of molecular events[29]. They can also be used to monitor feedback mechanisms of dorsal root ganglions during hyperglycaemia as it has been found that they increase their antioxidant activity during hyperglycaemia in anticipation of a further hyperglycaemic event[29].
Schwann cells have also been found to show similar results to dorsal root ganglions when exposed to hyperglycaemic conditions, however they are difficult to obtain due to the mass of other myelinated cells surrounding them[29]. They are also difficult to grow due to their lack of ability to attach to a substratum and do not fully represent intracellular activities involved in hyperglycaemia such as the increase of aldose reductase in the polyol pathway[29]. Schwann cell experiments have been replaced by experiments using transformed cell lines[29].
Transformed cell lines are easy to obtain and grow and form can also become electrically active by forming networks[29]. They can also make neurotransmitters for receptors they form and can therefore simulate a human internal environment[29]. Pheochromocytoma (PC12) cells are obtained from catecholamine-secreting adrenal tumours in rats and have been used extensively in research to show Bax protein involvement in mitochondria apoptosis, oxidative stress, ROS and many other implications following hyperglycaemia[29]. It has been found that differentiation of the PC12 cell line from their naïve to differentiated form yield obscuring data and therefore the appropriate level of differentiation must be completed[29]. The PC12 cell line would be a better model for analysing the effects of oxidative stress in diabetic neuropathy as they can differentiate in to mature cells and give a better representation of pathogenesis[29].
Aims
Following research, the aim of the experiment is to analysis the protective effects of vanillic acid against oxidative stress caused by hyperglycaemia. Vanillic acid will be used as the chosen antioxidant as it has been found to be one of the best phenolic acids to lower the effects of oxidative stress. An in vitro model using PC12 cells instead of Neuro-2A cells as adopted by Huang et al[23][28] will be used to represent human cellular mechanisms involved in hyperglycaemia. Using these cells will also make it easier to monitor molecular changes in the cells relating to oxidative stress and to measure time scales on which these events occur[29]. MG will be used as it has been found to be one of the most deleterious AGE-precursors to induce oxidative stress in diabetic neuropathy, therefore by exposing PC12 cells to MG it should give a good representation of human mechanisms. This is all in the aim to find an easily accessible and natural product, such as vanillic acid found in fruit and vegetables that can be used to alleviate the symptoms of diabetic neuropathy. A natural product would also avoid lengthy clinical trials and be able to be set to use for the increasing number of diabetic patients following the obesity epidemic[2].
I hypothesise that vanillic acid will increase the cell viability rate of methylglyoxal-exposed PC12 cells and reduce the effects of oxidative stress, compared to preliminary findings suggesting methylglyoxal reduces cell viability.
