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Dietary carbohydrate's such as monosaccharides such as glucose and galactose. Glucose is the most common carbohydrate currency of the body (Gaw et al., 2008) and is one of the most essential carbohydrates in biology. The importance of glucose becomes evidential in understanding that what it is used for, for example glucose is required for the production of adenosine-tri-phosphate (ATP) in glycolysis which is the initial stage of both aerobic and anaerobic respiration (Gaw et al., 2008) and (Rich, 2003) have both shown. The essentiality of glucose is also evidential in one of the later stages of aerobic respiration known as the citric acid cycle (TCA) (Rich, 2003).
According to (Tomlinson & Gardiner, 2008) Glucose concentrations are controlled via homeostatic mechanisms and the amount of glucose present within nerve tissue is always a result of a balance between input and output synthesis and catabolism. When the homeostatic mechanism fails this may result in a disease therefore, it is important that glucose concentrations are regulated. As according to (Anon., 2006) a fasting blood glucose concentration of greater than 7.7 mmol/litre is diagnostic of diabetes. In addition to this a normal range is found to be between 4 and 6 mmol/litre.
Diabetes Mellitus (DM) is a condition characterised by the inability to produce or respond to insulin, a hormone produced by the ¢-Islets of Langerhan cells in the pancreas (Anon., 2008). Type I DM (IDDM) affects 300,000 people in the UK, 20,000 of these being children under 15 (Bone, 2009). IDDM is an autoimmune condition mostly associated with juveniles, where by the patient's immune system is destroys the insulin-producing ¢-cells. On the other hand Type II DM (NIDDM) is known as a progressive condition as the body no longer responds to insulin and is associated with obesity (Anon., 2008).
As insulin is responsible for the uptake and metabolism of digestion products such as glucose and lipids, an insulin deficiency sequentially leads to hyperglycaemia and hyperlipidaemia, which consequently leads to other disease states such as atherosclerosis (Gleissner et al., 2007).
Hyperglycaemia in diabetes is said to cause up to a fourfold increase in the neuronal glucose levels (Gaw et al., 2008). If this is persistent, or if such episodes are chronic, then intracellular glucose metabolism leads to neuronal damage; this phenomenon is often referred to as glucose neurotoxicity (Tomlinson & Gardiner, 2008)
Complications of Diabetes Mellitus:
Diabetes mellitus (DM) has several complications which include diabetic retinopathy, diabetic nephropathy but with more relevance to this study diabetic neuropathy. Diabetic neuropathies (DN) are a heterogenous group of disorders are present a wide range of abnormalities (Pop-Busui et al., 2006) DN affects just over 50% of both IDDM and NIDDM sufferers with almost equal prevalence in both types, being slightly more common in NIDDM
The Polyol Pathway:
Figure 1, this shows examples of the pathways involved in the production of ROS (reactive oxygen species) which is then reflected in the overall increase of oxidative stress (Tomlinson, 2008)
There are several mechanisms associated with glucose neurotoxicity one of these being the polyol pathway (Chung, 2003) also sometimes referred to as the sorbitol pathway. (Chung, 2003), Suggests that this pathway was the first convincing hypothesis to emerge from the search for metabolic links between nerve dysfunction and hyperglycaemia.
The enzyme aldose reductase (AR) which is the first enzyme in the pathway has lower affinity for glucose than hexokinase hence most of the glucose in peripheral nerves is converted to glucose-6-phosphate during respiration and only trace amounts sorbitol can be detected (Tomlinson & Gardiner, 2008). The peripheral nerves take up considerable amounts of glucose in the absence of insulin. In diabetes it is likely that the hexokinase enzyme is 'saturated' and as a consequence the rise in glucose concentration brings it close to the binding with enzyme AR, as the affinity (Km) between glucose and AR in the presence of saturated hexokinase and increased [glucose] is sufficient for glucose to bind to AR (Lynch et al., 2000). However, Aldose reductase does not usually have a high affinity for glucose under normoglycaemic conditions but when excess glucose in hyperglycaemia is metabolised; it leads to intracellular sorbitol accumulation and a depletion of NADPH (Gleissner et al., 2007).
The pathway above is then initiated and for that reason appreciable amounts of sorbitol are formed and are not cleared as sorbitol has low plasma membrane permeability (Tomlinson et al, 2008).The accumulation of sorbitol results in direct tissue toxicity or to tissue swelling through an increased osmotic effect (Tomlinson & Gardiner, 2008). As (Tomlinson & Gardiner, 2008) state that sorbitol is understood to be a non-charged intracellular osmolyte. The synthesis of sorbitol therefore enables cells to buffer at high interstitial osmotic pressure. The combination between inappropriate intracellular osmolyte and sorbitol accumulation results in a reciprocal depletion of other cytoplasmic osmolytes, such as myoinositol and taurine. A 2008 study demonstrated that taurine administration to diabetic rabbits resulted in 30% decrease in serum glucose levels (Winiarska et al., 2008). Therefore, there is evidence to suggest that taurine has neuroprotective effects and the absence of osmolytes such as taurine, results in hyperglycaemia and diabetic complications.
Schwann cells are a variety of glial cell that keep peripheral nerve fibres (both myelinated and unmyelinated) alive (Jessen & Mirsky, 2005). They are significantly rich in aldose reductase therefore they may play a role in the development of DN.
On the other hand there are other mechanisms of glucose neurotoxicity to consider in the pathogenesis of neuropathy. There is a prospect that increased AR activity or increased flux through the pathway affects other signalling systems such as p38 MAP kinase a class of mitogen-activated protein kinases which are responsive to stress stimuli such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in cell differentiation and apoptosis (Cooper, 1997).
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. As a result of intracellular hyperglycaemia, pre-cursors to AGEs, dicarbonyls, are formed during the oxidation of glucose to glyoxal. These dicarbonyls react with the amine groups on intracellular proteins and form AGEs. Methylglyoxal (MG) is a highly potent dicarbonyl and increases sensitivity of cells to damage. 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. Those cells with RAGE are particularly associated with atherosclerosis and so elevated AGE levels have been linked with the increased incidence of cardiovascular disease in diabetic patients.
AGEs interact with RAGE and causes activation of mitogen-activated protein kinases (MAPK), protein kinase C and nuclear factor kappa B (NF-«B). NF-«B translocates to the nucleus where its p65 subunit up-regulates transcription of adhesion molecules and pro-inflammatory molecules such as ICAM, IL-6 and TNF. NF-«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. Damage occurs by macrophages phagocytosising the nerve myelin and AGEs modifying the intracellular matrix proteins; tubulin, neurofilament and actin, by cross-linking them. 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.
This is promoted by glucose though a combination of free radical generation and impaired free radical scavenging. Hydrogen peroxide is produced by the action of superoxide dismutase on superoxide (O2-), which is itself generated by increased oxidative metabolism of glucose in the mitochondria. The sorbitol pathway compromises the glutathione (GSH) cycle by consuming the proton donor NADPH. This reduces the capacity of glutathione peroxidase to metabolize hydrogen peroxide to water, and as a result increases the passage of hydrogen peroxide into Fenton reaction production superhydroxyl radicals (.OH). The ratio of GSH/GSSG is a definitive indicator of the redox state of the cell. Evidence of this implicating oxidative stress from the polyol pathway comes from an investigation whereby diabetic rats with an aldose reductase inhibitor which normalised the ratio from its unbalanced state in untreated diabetics (Pop-Busui et al, 2006).
Hyperglycemia is reported to induce oxidative stress through multiple pathways such as redox imbalances secondary to enhanced aldose reductase (AR) activity, increased advanced glycation end products (AGE), altered protein kinase C (PKC) activity, especially Î²-isoforms, prostanoid imbalances, and mitochondrial overproduction of superoxide. These pathways all converge in the production of oxidative stress. This has been documented in peripheral nerve, dorsal root and sympathetic ganglia system, and may contribute to nerve blood flow and conduction deficits, impaired neurotrophic support, changes in signal transduction and metabolism and morphological abnormalities characteristic of DN.
The reactive species (free radicals) can be divided into different categories' e.g. reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive chlorine species (RCS). Their most prominent members include superoxide (O2-), hydroxyl radical (OH-), peroxyl radical (ROO-) in the ROS group, and nitric oxide NO in the RNS group (Pop-Busui et al, 2006).
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. Donated electrons are then transferred through the membrane complex in the mitochondria and join oxygen to form water as a by-product. 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. 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}.
Methylglyoxal (MG) on neuronal (PC12 cells) survival by determining the link between PC12 survival and MG and whether or not
Methylglyoxal is a significant AGE precursor therefore being a popular candidate for research into oxidative stress in diabetic neuropathy. Methylglyoxal (MG) is formed as a result of high intracellular levels of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate from hyperglycaemia. MG has been found in high concentrations in diabetic patients and this also corresponds with patients with diabetic complications. 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. 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. Therefore when glutathione activity is reduced due to sorbitol accumulation and NADPH depletion, the detrimental effects of MG can't be neutralised.
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. This as a result activates endothelial cells including in the endoneurium which is a thin layer of endothelial cells enveloping each peripheral nerve fibre. 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. Increased ROS concentrations in the endothelium surrounding the neurones lead to oxidative stress, demyelination and neuronal apoptosis.
Neuronal apoptosis caused by ROS and oxidative stress specifically describes the death of Schwann cells surrounding the neurone. Schwann cells surround peripheral neurones and provide insulation which is important for conduction of electrical impulses and are crucial for neuronal survival. The condition of diabetic neuropathy encompasses the damage and death of Schwann cells leading to the loss of feeling in peripheral areas. 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.
Given that the imbalance between increased production of free radicals and the antioxidants present leads to oxidative stress caused by glucose toxicity, one way to combat oxidative stress is by increases the amount of antioxidant present. According to (Vincent et al, 2005) short term hyperglycaemia produces oxidative damage and apoptosis in neurons. If rapid development of oxidative stress is a mediator of the death of Dorsal root ganglia (DRG) neurons, then antioxidants may protect DRG neurons during a hyperglycaemic insult. EGCG (Epigallocatechin-3-gallate) is just one of several antioxidants which inhibit ROS activity by either the destruction or protection from free radicals (Cherbonnel-lasserre C et al, 1997). According to (Levites et al, 2003) 'green tea extract and its main polyphenol constituent (-)-EGCG possess potent neuroprotective activity in cell culture. The central hypothesis guiding this study is that EGCG may play an important role in amyloid precursor protein (APP) secretion and protection against toxicity induced by Î²-amyloid (AÎ²) (Levites et al, 2003). The present study shows that EGCG enhances the release of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPÎ±) into the conditioned media of human SY5Y neuroblastoma and rat pheochromocytoma PC12 cells.
Inhibition of protein kinase C (PKC) with an inhibitor, or by down regulation of PKC, blocked the EGCG induced sAPPÎ± secretion, suggesting the involvement of PKC. PKC is recognised as one of the enzymatic pathways of glucose metabolism which initiates a stress responses reflected in the overall increase of oxidative stress (Vincent et al, 2005). EGCG is not only able to protect, but it can rescue PC12 cells against the AÎ² toxicity in a dose dependent manner. There is a possibility that this mechanism is very similar to that of methylglyoxyl (AGE's) induced oxidative stress.
The aim of the investigation is to illustrate the effect of methylglyoxyl (MG) on neuronal (PC12) survival by determining the link between PC12 survival and MG and whether or not the antioxidant EGCG can either slow the deleterious effects of MG or prevent cell degradation (Gomes et al, 2009). The molecular outcomes of raised glucose levels can be indirectly assessed via methylglyoxyl and generally be examined in neuronal cultures. It can then be further verified in animal models of diabetes. This report identifies the literature behind the investigation via the assessment of neuronal cultures subjected to these conditions.
Materials and Methods:
PC12 cells were obtained from (ATCC). Cells were maintained in RPMI (Sigmaâ€Aldrich), supplemented with 10% (v/v) heatâ€inactivated horse serum (Invitrogen) and 5% (v/v) fetal bovine serum (Invitrogen) at 37°C in a 90% humidified atmosphere containing 5% CO2. The concentration of glucose in the RPMI medium was 4.5mg/ml. According to the manufacturer's instructions, PC12 cells were maintained with culture medium containing 4.5 mg/ml glucose.
PC12 cells were plated in Nunc dishes coated with laminin (20 Î¼g/ml, Sigma, UK) and poly DLâ€ornithine (0.5mM, Sigma, UK) under serum free conditions in RPMIâ€1640 medium (Sigma). NGF (2ng/ml, Sigma, UK) was added to the cells to promote neuronal differentiation of the PC12 cells. After the initial 72h postâ€plating, PC12 cells exhibited neurite outgrowth greater than 2 cell body diameters indicating neuronal differentiation. The cells were then treated and incubated with varying methylglyoxal concentrations ( mM, Sigma, UK) and assayed at varying timeâ€points of 24 h, 48 h and 72 h to determine the suitable incubation period and concentrations which reduced cell viability to approximately 50%. All experiments were carried out in replicates for reproducible results.
MTT cell viability assay:
Cell viability, based on mitochondrial dehydrogenase, was determined by MTTâ€formazan assay after treatment with the varying methylglyoxal concentrations and incubation at varying timeâ€points. The assay is based on the ability of living cells to reduce MTT solution to insoluble formazan (when incubated for approximately 1hour at 37°C); therefore the amount of formazan produced is proportional to the number of living cells, and is measured at 570nm using an ELISA microplate reader. Results were also carried out in replicates to minimise experimental errors.
All results evaluated using mean ± SEM. Significance of difference between multiple group means was also tested using oneâ€way ANOVA, with a probability level of P<0.05 considered to be significant.