The Effects Of Methylglyoxyl On Pc12 Cells Biology Essay

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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.


PC12 are grown in vitro via NGF (nerve growth factor) which promotes PC12 survival (Kawaja et al 1992). Once the cells have differentiated they can then be treated with MG. In order to find the optimum MG concentration three different concentrations are used, they are as follows 1µm, 100 µm, and 1000 µm. These are based on the concentrations of MG found within diabetic patients and patients with diabetic neuropathy. In order to see the effect of the antioxidant EGCG, MG should be allowed to show its cytotoxic effect (Godbout et al ,2002) therefore it is required that the most appropriate time for MG induced oxidative stress initially be found. If the cells are reduced to approximately 50% cell viability then the effect of the addition of the antioxidant can be seen.


PC12 cells treated with MG are likely to show some form of cell degradation as MG is understood to have cytotoxic properties (Kim, et al 2009) therefore, PC12 treated with MG is expected to show cellular injury (evidence). Hence, it is expected that there will be a reduction in the number of PC12 seeded. Furthermore when PC12 are treated with antioxidant EGCG the amount cell should show increased viability (Vincent et al, 2005)


Dietary carbohydrate is digested in the gastrointestinal tract to simple monosaccharides. Glucose is the most common carbohydrate currency of the body. Glucose concentrations are controlled via homeostatic mechanisms the level 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.

Diabetes mellitus an endocrine disorder is found where the body's is unable to control glucose concentrations and therefore diabetics show increased levels of glucose known as hyperglycaemia (GAW, 2008). Hyperglycaemia in diabetes causes up to fourfold increases in neuronal glucose levels. If this is persistent, or if such episodes are regular events, then intracellular glucose metabolism leads to neuronal damage; this phenomenon is often referred to as glucose neurotoxicity (Tomlinson et al, 2008).

Glucose is a very important carbohydrate in biology as the living tissue cells use glucose as a source of energy via aerobic respiration (Fraser, 2002) . Glucose is required for the production of ATP (adenosine-tri-phosphate) in glycolysis as well as in the TCA cycle (Rich, 2003).

Insulin is the principal hormone affecting blood glucose levels and an understanding of its actions is an important prerequisite to the study of diabetes mellitus (Gaw et al 2008). Insulin is a small protein that is synthesised by the beta cells of the islets of Langerhans of the pancreas. Insulin secretion causes a decrease in glucose concentration by converted glucose into other products such as glycogen which can be stored in the liver and released and converted back during hypoglycaemia (GAW, 2008). Glucose can also be stored in muscles and fat (adipose tissue). Insulin is known to activate the GLUT4 inward glucose transporter in muscle and fat and promotes phosphorylation of glucose in the liver. The body can then store fat in adipocytes and glycogen in the liver which are maintained. The high glucose demand of muscles can be met from immediately available short-term glycogen stores. This process handles large amounts of glucose while keeping plasma glucose concentration in a relatively narrow window (Hendenreich et al, 1989).

The inability of an individual to either respond to the presence of insulin often results in diabetes and glucose neurotoxicity as a serious consequence of long term diabetes and furthermore the clinical syndrome is known as diabetic neuropathy (Anon, 2008).

The end stage of this condition is loss of protective sensation, particularly in the feet, which makes the sufferer vulnerable to insensible trauma. Such occurrences as well as poor wound healing occur in poorly controlled diabetes), can lead to foot ulcers and gangrene and are major causes of amputation.

Diabetics not only show increased glucose concentrations but also show increased methylglyoxyl (MG) concentrations. MG arises from glucose metabolism along the advanced Glycation pathway as shown by figure 1 below therefore assessing MG changes concentrations is an indicator of the severity of the disease in diabetic patients MG concentrations are higher and even higher in patients with further complications of diabetes. (Bourajjaj et al, 2006)

Diabetes mellitus (DM) has several complications which include diabetic retinopathy, diabetic nephropathy but with more relevance diabetic neuropathy. Diabetic neuropathies (DN) are a heterogenous group of disorders and present a wide range of abnormalities (Pop-Busui et al 2006). Diabetic neuropathy can be further sub-divided into diabetic peripheral neuropathy (DPN) and (DAN) diabetic autonomic neuropathy. DPN's is understood to be a significant cause of severe pain, suffering, and disability and accounts for the majority of nontraumatic lower extremity amputations (Pop-Busui, et al 2006). Diabetic autonomic neuropathy may affect any sympathetic or parasympathetic function with life threatening consequences such as sudden cardiac death and silent myocardial ischemia.

Considering that the epidemic explosion of diabetes throughout the world and that DN's are most among the most common long term complications of diabetes, they are a significant cause of morbidity and mortality. But unfortunately, to date, besides a tight glycaemia control, a viable treatment for human DN is not available. Therefore understanding its pathogenesis is critical for a better management.

The development of vascular complications of diabetes begins with an underlying genetic predisposition which when acted on by various initiating events, results in inflammatory changes that may precede hyperglycemia. Inflammation and hyperglycaemia unleash a cascade of events that can affect cellular proteins, gene expression and cell-surface receptor expression in the endothelium, ultimately resulting in progressive pathologic changes and subsequent vascular complications such as some of the symptoms seen within DN (Pop-Busui et al 2006).

However although increasing evidence provides support for a micro-vascular basis of DN, its pathophysiology is still poorly understood (Pop-Busui et al, 2006). This review therefore aims to illustrate with the use of evidence that oxidative stress plays an important role in the onset and in the development of DN.

In order for glucose to be delivered to the CNS neurons it must cross the blood-brain barrier however the brain as part of the nervous system preferentially uses glucose as an energy source and is not metabolically adjusted for the metabolism of free fatty acids.

The brain contains microvessels which express the transport GLUT1 , which is unresponsive to insulin hence brain glucose uptake appears to be independent of insulin action. Lower expressions of GLUT4 and GLUT1 are found within the lateral hypothalamus and the arcuate nucleus and the globus pallidus. mRNA coding for accessory molecules e.g. glucokinase which is involved in the phosphorylation of glucose and the insulin receptor are also expressed in some of the brain regions, but insulin regulated glucose uptake has not yet been proven to occur in the brain. When neurons are stupdided in culture, thus removing any influence of a vasculature-interstitium barrier glucose uptake has been shown to be independent of insulin. GLUT4 is regionally distributed throughout the body suggesting that insulin dependent glucose uptake might occur in specialised neuronal phenotypes. Vascular barriers are the only protection against glucose toxicity during hyperglycaemia for neurons (Tomlinson et al, 2008).

According to (Tomlinson et al, 2008) another glucose transporter GLUT8 has been identified in the brain but more specifically in the hippocampus, the cerebral cortex and the hypothalamus. Studies of the transporter within the hippocampus suggests that it does not respond to the presence of insulin but is activated by the presence of glucose itself, which causes GLUT 8 to migrate towards the plasma membrane therefore GLUT8 may represent an insulin-independent glucose uptake carrier that is specific to certain neurons. However queries still exist around subject as alternatively GLUT8 initiation maybe due to increases in intracellular glucose that occur when glucose is liberated from oligosaccharides.

Some apparent specializations of glucose sensitivity might enable neurons to respond to glucose in order to fulfil their physiological role as part of the appetite-satiety system of the brain the GLUT8 mechanism may perform these actions however some CNS neurons (especially those in the arcuate nucleus) also express glucokinase which is the glucose sensor that is expressed in the β-cells of the pancreatic islets. These neurons are also found to co-express the apetite modulating peptide neuropeptide Y (NPY), further suggesting that they might have a role in feeding behaviour. Many of the neurons also express KAtp channels which shows that glucose can regulate the excitability, as in the β cells. The β cells raised intracellular glucose promotes ATP synthesis which leads to the internal blocking of ATP sensitive K+ channels hence reduced K+ flux, increased excitability and the stimulation of insulin release. The precise interactions between glucose-sensing, ion fluxes and neuropeptides still requires clarification (Tomlinson et al, 2008).

Evidence suggests that the blood-brain barrier offers some protection to the brain, but evidence suggests that this operates less effectively for peripheral nerves, because hyperglycaemia is associated with marked increases in intracellular glucose (Tomlinson et al, 2008). Furthermore more specialised cells of the brain have refined glucose affinities, but it is not known whether this makes them more or less vulnerable to glucose toxicity. The type of neuronal cells used within this experiment are PC12 which is a noradrenergic cell line derived from a rat pheochromocytoma (Greene, 1978). In culture medium PC12 cells undergo mitosis when nerve growth factor is included in the medium

Molecular mechanisms of glucose neurotoxicity

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. This pathway was the first cogent hypothesis to emerge from the search for metabolic links between nerve dysfunction and hyperglycaemia. Studies on the eye lens verified this link. The pathway is shown below:

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) C:\Users\Theo\Pictures\2010-02-22\001.jpg

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. 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) of 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).

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. Sorbitol is understood to be a non-charged intracellular osmolyte. The synthesis of sorbitol enables cells to buffer high interstitial osmotic pressure. The combination of inappropriate intraceullular osmolyte and sorbital accumulation results in a reciprocal depletion of other cytoplasmic osmolytes, such as myoinositol and taurine as expression of their uptake carrier protein is regulated by intracellular osmolarity at the transcription level. Schwann are significantly rich in aldose reductase therefore it might be the schawann-cell derived input to the pathogenesis of neuropathy. On the other hand there are other mechanisms of glucose neurotoxicity to consider in the pathogenesis of neuropathy so this requires further investigation. There is the prospect that increased AR activity or increased flux through the pathway affects other signalling systems therefore this is an important issue when considering the pathogenesis of neuropathy. There is said to be a clear interaction with p38 MAP kinase but other links are yet to be determined.

Oxidative Stress and Excessive free radical production

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 cycle by consuming the proton donor NADPH. This reduces the capacity of glutathione peroxidise 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).

Oxidative stress refers to the situation of severe imbalance between the production of free radicals and the antioxidants defence mechanisms, leading to potential tissue damage. Free radicals are a group of highly reactive species hence in complications of diabetes such as DN the increased ratio of free radicals leads to an ineffectiveness antioxidant present.

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 β-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).

Protein Glycation and intracellular signals

Another consequence of hyperglycaemia is the non-enzymatic Glycation of proteins. The first reaction of this type to be described was the formation of a Schiff base by the direct addition of open chain glucose to lysine groups on proteins. The Schiff base undergoes a slow, spontaneous rearrangement formally known as the Amadori rearrangement. The rearrangement results in the formation of a stable advanced Glycation end-product (AGE). The RAGE ligand carboxymethyllsine has recently been shown to activate nuclear factor κB (NFκB) signalling in neurons which is involved in responding to stimuli associated with stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens (Perkins,2007).

In addition the importance of fast AGE formation from glucoxidation products such as MG, widens the possibilities for causing dysfunction. It is said to be possible that through RAGE activation, AGE's act by disrupting intracellular signals and that reactive Glycation products in cells can destabilize biological processes (Tomlinson et al, 2008). However further research is needed in this area as it is not exactly completely clear how this process works. Structural and functional assessment of the impact of Glycation on nerves has been hampered by the lack of molecules that specifically inhibit Glycation reactions (Tomlinson et al, 2008). The molecule 'Aminoguanidine' has been used to this end but, owing to its other pharmacological properties it's found to inhibit nitric oxide synthase and monoamine oxidase and therefore used as an investigative tool in vivo is limited.

Development of new Glycation inhibitors is therefore essential for further understanding of protein Glycation. There are a few new Glycation inhibitors such as pyridoxamine which might offer new insight into the effects of macromolecule Glycation in nerve dysfunction (Tomlinson et al, 2008).

MAP kinases are activated by high extracellular glucose levels. This is said to be true in primarily neurone cultures, diabetic rats and even sural nerve specimens from diabetic patients. Given the responsiveness of the MAP kinase c-jun N terminal kinase (JNK) and p38 to stress, this activation might arise from hypertonicity, oxidative stress, RAGE activation or a combination of these effects.

It has been suggested on the basis of experiments in cultured embryonic neurons that this causes neuronal apoptosis. However, it is well established that adult neurons survive JNK activation by expressing a truncated form of p73 that does not propagate the death signal and therefore no evidence of neuronal apoptosis in rats with long term diabetes. This happens regardless of the activation of capase-3 which leads to apoptosis (Tomlinson et al, 2008). Activated JNK might however compromise the axonal cytoskeleton by directly hyperphosphorylating neurofilament proteins which may contribute to long term axonal shrinkage in diabetic neuropathy (Tomlinson et al, 2008).

Astrocytes which are glial cells located in the brain and spinal cord are known to be susceptible to glucose-triggered oxidative stress. This leads to subsequent damage and cell death (Tomlinson et al, 2008). However the confusion arises with whether these mechanisms operate in vivo during hyperglycaemia as the blood-brain barrier offers protection against glucose toxicity. Nevertheless, if the barrier is damaged during a cerebral infarct, glucose toxicity then becomes a serious threat. It is well-documented that the severity of strokes in diabetic patients is greater than in healthy individuals (Tomlinson et al, 2008).

Deficient neurotrophic support has well-defined effects on gene expression and function in sensory neurons (Fernyhough, 1995). The effects of glucose on neurotrophin production by neuronal targets keratinocytes or myocytes have not been reported. However, production of NGF by cultured Schwann cells is inhibited by raised glucose levels (Tomlinson, 2008). This can be covered by the addition of an aldose reductase inhibitor to the medium there is further evidence that says that the inhibition of Schwann cell NGF production is due to oxidative stress and that treatment with an antioxidant such as EGCG in this case normalizes NGF levels.

Antioxidant EGCG

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 β-amyloid (Aβ) (Levites et al, 2003). The present study shows that EGCG enhances the release of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPα) 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 sAPPα 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 Aβ 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.


MG can modify tissue proteins through the Maillard reaction, resulting in advanced glycation end products (AGEs), which can alter protein structure and functions. Several MG-derived AGEs have been described, including argpyrimidine, which is a fluorescent product of the MG reaction with arginine residues. Scientists detected significant amount of argpyrimidine in rat kidney mesangial cells cultured in media containing high concentrations of glucose. Heat shock protein 27 (Hsp27) was identified by liquid chromatography tandem mass spectrometry as a major anti-argpyrimidine immunoreactive protein. We confirmed this finding by reciprocal co-immunoprecipitation and by Western analysis. Diabetic rats contained more argpyrimidine-modified glomerular Hsp27 than non-diabetic animals. Additional studies showed that MGO-induced modification of Hsp27 decreased its binding to cytochrome c. Our results suggest that Hsp27 is a major target for MGO modification in mesangial cells (Padival et al, 2003).

It is important to recognise the link between MG and Aβ as the use of antioxidant EGCG is said proven to protect against toxicity induced by Aβ. Diabetics exhibit elevated levels of carbohydrates and their oxidized by products such as methylglyoxyl (MG). With the excess production and accumulation of the Aβ peptide in Alzheimer's disease (AD), the linkage between diabetes and Alzheimer's disease may involve Aβ and MG (Michelson et al, 2008). Studies on the effects of MG induced Aβ modifications show that MG affected the rate of Aβ aggregation as compared with a control, indicating that AGEs may influence Aβ packing densities. These observations therefore suggest a plausible explanation for the relationship between diabetes and AD but more importantly suggests that Aβ packing densities may have an association with oxidative stress and with the addition of EGCG according to (Vincent et al, 2005) PC12 may show increased cell viability.