Methylglyoxal Nonenzymatic Mg Formation Biology Essay

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Methylglyoxal (MG) was first chemically characterized in 1885 by the German researcher, Dr Baumann (Banumann 1885). Since then, MG is known to be inevitably produced as a byproduct of sugar, fat, and protein metabolism, and can be found in virtually all mammalian cells (Kalapos 2008). Indeed, MG, is implicated along with its adducts, advanced glycation endproducts (AGEs) and ROS, are involved in normal physiology functions, such as cellular transduction systems, including ERK 1/2 (Du et al. 2003; Blanc et al. 2003), c-Jun N-terminal kinases (JNK) (Kyriakis and Avruch, 1996; Du et al. 2000), and p38 MAPK pathways (Kyriakis and Avruch 1996), tissue remodeling maintenance and normal functions of the primary immune response (Di Loreto et al. 2004). However, overproduction of MG could result in endothelial dysfunction (Wu and Juurlink 2002), wall inflammation, and vasoconstriction (Pedchendo et al. 2005); thus leading to several insulin resistance diseases, such as hypertension (Wang et al. 2004, 2005, 2008; Vasdev et al. 1998a, b; Wu and Juurlink 2002; Wu 2006; Tomaschitz et al. 2010) and diabetes mellitus (Wang et al. 2007; Riboulet-Chavey et al. 2006; McLellan et al. 1994).

As a member of the reactive carbonyl species (RCS), MG is formed mainly through the fragmentation and elimination of phosphate from the triosephosphates: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P) (Wu 2006; Figure 3.1-1). The triosephosphate pool, in turn, is regulated by both glycolytic and gluconeogenic pathways. For example, upon elevated levels of plasma glucose levels, per se 100 mg/dl or 5.6 mM, this would enhance cellular glycolysis and thus MG generation (Chang and Wu 2006; Wu 2006), whereas, upon enhanced gluconeogenesis, during starvation periods, it could be quite likely that MG production would be lessened. Although it is currently unknown how MG enters the cell, by increasing available glucose in cultured cells, such as human red blood cells, bovine endothelial cells, or aortic smooth muscle cells, MG levels are significantly elevated (Thornalley 1988; Thornalley 1996; Wang et al. 2006). Indeed, by increasing glucose, MG-induced ROS (Dhar et al. 2010; Wang et al. 2006), as well as MG-induced AGE formation are also increased (Thornalley 2003).

3.1.2 Enzymatic formation

This dicarbonyl molecule can also be generated by lipolysis, which uses acetone as a precursor, (Casazza et al. 1984; Koop and Casazza 1985), or by the metabolism of threonine (Ma et al. 1989) or aminoacetone (Lyles & Chalmers, 1992) from protein catabolism (Figure 3.1-1). MG derived from acetone is synthesized by acetal monooxygenase by amino oxidase (AMO) (Koop and Casazza 1985). In protein catabolism, semicarbadize-sensitive amine oxidase (SSAO) catalyzes the conversion of aminoacetone to MG (Casazza et al. 1984; Lyles and Chalmers 1992), where aminoacetone was shown to be significant source of MG formation in A-10 cells (Dhar et al. 2010). Currently, two forms of SSAO are known: soluble form, typically in the blood stream, or membrane-bound, for example on VSMCs (Ekblom 1998; Tressel et al. 1986). Indeed, significantly elevated serum SSAO activity levels were found in patients with diabetic complications, including retinopathy or nephropathy (Yu et al. 2003), although, with unclear mechanisms (Wu and Juurlink 2002).

3.2 Metabolism of MG

3.2.1 Glyoxalase system

MG is a reactive intermediate that is inevitably produced under normal conditions; therefore, naturally, there is a MG detoxification pathway. The degradation of MG occurs mainly by the ubiquitous glyoxalase system, which is present in the cytosol of all mammalian cells (Chang and Wu 2006). This MG detoxification system consists of two enzymes, glyoxalase-I (Gly-I) and glyoxalase-II (Gly-II), as well as the cofactor, GSH (Chang and Wu 2006) (Figure 3.1-1). The process involves the irreversible conversion of MG to S-D-lactoylglutathione, via Gly-I activity and GSH as a cofactor, then to D-lactate by Gly-II activity. At this stage, D-lactate can be further converted to pyruvate, which can either be metabolized in gluconeogenesis or enter the citric acid cycle (Chang and Wu 2006).

3.3 Cellular toxicity of MG

The net output of MG levels in a cell is the summation of its generation and degeneration. However, as a consequence of overconsumption of foods high in carbohydrates, fat, or beverages containing high amounts of ethanol, or coffee for that matter, could upset this delicate balance, leading to an accumulation of MG. As such, this can produce deleterious effects.

3.3.1 Production of reactive oxygen species (ROS)

The generation of free radicals and ROS are needed for normal physiology functions, such as cellular redox signaling, tissue remodeling maintenance, and normal functions of the primary immune response (Yan et al. 2006; Chang and Wu 2006; Wu 2006). However, overproduction of free radicals and ROS contributes to the development of endothelial dysfunctions, wall inflammation, and vasoconstriction; thus leading to several insulin resistance diseases, such as hypertension, atherosclerosis, and diabetes mellitus (Chang and Wu 2006; Wu 2006: Kalapos 2008). Oxidative stress is characterized when there is an imbalance between oxidants and anti-oxidants, which could be from an increased production and/or decreased degradation of ROS (Chang and Wu 2006).

Indeed, MG is also known to generate ONOO-, H2O2 and superoxide anion (O2.-) via nonenzymatic reactions (Chang et al. 2005). ONOO- is formed when O2.- interacts with NO, which also decreases the bioavailability of NO (Chang and Wu 2006). Indeed, MG induced a concentration-dependent increase in ROS/reactive nitrogen species (RNS) in A-10 cells, which was attenuated by GSH and N-acetyl cysteine (NAC), a MG scavenger, superoxide dismutase (SOD; a O2.- scavenger), and diphenyliodonium (DPI; a NADPH oxidase inhibitor) (Chang et al. 2005). Furthermore, it was also demonstrated that MG can enhance NADPH oxidase-mediated production of H2O2 in rat kidney mesangial cells, which was abrogated by SOD (Ho et al. 2007), as well as enhance H2O2 levels in rat hepatocytes (Kalapos et al. 1994). Moreover, MG administration induced oxidative stress in isolated VSMCs from SHRs, due to significantly higher levels of GSSG, and significantly lower levels of GSH and the anti-oxidant activities of glutathione reductase (GSSG-Red) and glutathione peroxidase (GSH-Px) with comparison to the wild type cells (Wu and Juurlink 2002). Additionally, it was demonstrated that MG can enhance H2O2 levels in neutrophils (Ward and McLeish 2004), and induces platelet H2O2 accumulation and aggregation (Leoncini and Poggi 1996).

3.3.2 Interaction with anti-oxidant enzymes

Because MG is a reactive carbonyl, it can directly interact with anti-oxidant enzymes, such as GSH-Px (Paget et al. 1998) and GSSG-Red (Blakytny et al. 1992), via glycation. Normally, GSH-Px scavenges H2O2, by using GSH as a cofactor, which is then oxidized to GSSG (Desai et al. 2010). GSSG-Red then reduces 1 mole of GSSG to form 2 moles of GSH, thus replenishing cellular GSH levels (Desai et al. 2010). However, this MG-induced impairment of the GSH recycling system, involving the main enzymes GSSG-Red and GSH-Px, could shift the balance of oxidants and anti-oxidants to higher levels of oxidants, thus leading to oxidative stress (Desai et al. 2010). This deleterious phenomenon would impair the detoxification of MG, thereby enhancing its half-life and pro-oxidant potential.

3.3.3 Modification of protein

Due to its electrophilic nature, MG can readily react with specific arginine, lysine, or sulfhydryl residues in enzymes, lipids, DNA, and receptors (Chang and Wu 2006; Wu 2008). As a result, this will lead to alternations in biological functions in VSMCs and ECs located within the blood vessel walls (Wu 2008). As the most reactive AGEs precursor, MG can undergo an irreversible glycation reaction on the targeted protein to yield AGEs (Chang and Wu 2006; Wu 2008). These highly reactive species will either directly interact with other cellular proteins and/or nucleic acids, or with their receptors (RAGE). RAGE signal transduction mechanisms are known to greatly induce oxidative stress (Wu 2008). Higher levels of MG precursors, such as glucose and fructose (Wu and Juurlink 2002), can lead to higher levels of MG and MG-generated AGEs.

Since MG can readily react with arginine or lysine residues in proteins, leading to glycation, this could also irreversibly inhibit an enzyme activity. Arginine and lysine are common occurring amino acids in the catalytic active sites of enzymes (Wu and Juurlink 2002). As mentioned previously, MG can increase oxidative stress by inactivating GSH-Px and GSSG-Red via glycation (please see section 3.3.2). Likewise, Wu and Juurlink (2001) showed that GSH expression and the activities of GSSG-Red and GSH-Px were lower in VSMCs from hypertensive rats. Thus, this suggests a link between MG-induced AGE formation to decreased GSH expression.

Interestingly, these glycation reactions with the selected amino acid residue on the targeted protein are highly selective (Chang and Wu 2006). For example, the reaction of MG with arginine produces hydroimidazolone Nε-(-5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (Ahmed et al. 2003) and argpyrimidine (Ahmed et al. 2002), whereas the irreversible reaction of MG with lysine leads to the formation of Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL). Indeed, MG-induced AGE formation, including CEL, are labeled as indicators of carbonyl overload in vivo (Singh et al. 2001), and they are also connected to age (Ando et al. 1999; Li et al. 1996). At the cellular levels, the deleterious effects of MG-induced AGE formation would lead to the inactivation of enzymes, receptors, protein carriers, and structural proteins (Chang and Wu 2006); whereas clinically, AGE production have been implemented to the development of neuropathy, retinopathy, and nephropathy in diabetic patients (Sugiyama et al. 1996).

3.3.4 Modification of nucleic acids.

Not only does MG react readily with arginine or lysine amino acid residues on proteins, but it can also react with guanyl residues in DNA and RNA strands. This can in fact lead to translational, as well as transcriptional abnormalities within a cell (Thornalley 1996). However, more research is needed in this field.

3.3.5 Pro-inflammatory agent

In cardiovascular diseases, one of the major effects of oxidative stress is the induction of pro-inflammatory molecules (Ogata et al. 2000). Indeed, NF-κB plays a key role in regulating cell survival, as well as the immune response to infection. Upon activation of NF-κB, by pro-inflammatory cytokines (TNF-α, or IL-1), it will induce pro-inflammatory responses by promoting the expression of genes that mediate inflammatory reactions, such as adhesion molecules (inter-cell adhesion molecule-1; ICAM-1 and vascular cell adhesion molecule-1; VCAM-1), and other cytokines (IL-8, TNF-β) (Ogata et al. 2000; Marumo et al. 1997). Thus, it is not a surprise to know that NF-κB has been linked to diseases where inflammation is an issue, including insulin resistance diseases and atherosclerosis (Marumo et al. 1997).

In light of this, MG-induced ROS and AGE generation can play an important role in activation of NF-κB. For an example, by degrading IκBα (inhibitor of κB), it was shown by Wu and Juurlink (2002) that MG-induced H2O2 can activate NF-κB p65 in VSMC in SHRs. It was also observed that NF-κB can be activated by O2.- and H2O2 (Canty et al. 1999; Ogata et al. 2000), as well as ONOO- (Cooke and Davidge 2002) in endothelial cells. Additionally, it was further demonstrated by Wu et al (2004) that significantly higher expression levels of NF-κB occur in the kidney of SHRs. Likewise, MG administration elicit the activation of NF-κB p65 in cultured rat VSMC and from aorta (Wu and Juurlink 2002) and mesenteric artery (Wu 2005), where in both studies, NAC significantly decreased the MG-induced inflammatory responses. Lastly, it was shown that MG administration significantly increased both the transcriptional and translational expression of nervous growth factor, as well as IL-1β, in hippocampal neural cells (Di Loreto et al. 2004). Thus, it is strongly agreed that overproduction of MG can elicit pro-inflammatory responses in the vasculature.

3.4 The association of MG and diseases

At normal physiological levels, MG regulates signal transduction systems and various homeostatic mechanisms of cellular functions, balances redox reactions, as well as influence cell survival. However, abnormally high levels of MG and MG-induced production of ROS and AGEs are implicated in the alternation of vascular reactivity, wall inflammation, and endothelial dysfunction. The Western diet (foods high in carbohydrates and/or fats, as well as beverages with high amounts of sugar), as well as lack of exercise, does not help the situation. Numerous studies have linked high levels of MG and MG adducts in the impairment of the cardiovascular system, resulting in diabetes, hypertension, heart disease and stroke, which are the number killers in North America (WHO 2007).

3.4.1 Hypertension

High MG levels are associated with the development of high BP in SHRs and may be a causative factor for the development of hypertension in this model (Wang et al. 2004, 2005, 2008; Vasdev et al. 1998a, b; Kamencic et al. 2000). Indeed, it was shown that Wistar Kyoto (WKY) rats treated with MG (0.2% to 0.8%) (Vasdev et al., 1998a) or fructose (4%) (Vasdev et al. 1998b), a precursor of MG (in drinking water), displayed a continuous increased in systolic BP. Indeed, the WKY rats that were treated with both MG and NAC, a MG scavenger, did not development high BP (Vasdev et al. 1998a). These authors also found that the MG treated rats showed smooth muscle cell hyperplasia in the small artery and arterioles of the kidney, where the MG+NAC treated rats showed no such change (Vasdev et al. 1998a). VSMC proliferation is one of the characteristics of hypertension (Irani 2000). The hyperplasia in the resistance arteries may be due to the cell proliferative effects of MG, since MG can activate both ERKs (Du et al. 2003; Blanc et al. 2003) and NF-κB p65 pathways in VSMCs (Wu and Juurlink 2002). These pathways promote cellular proliferation and survival (Chang and Wu 2006). Proliferation and apoptosis of VSMCs are important cellular events of vascular remodeling (Yang et al. 2004); however, over stimulation of these pathways severely effects vascular integrity (Figure 3.4.1-1).

Additionally, Wang et al (2004, 2005) demonstrated the plasma MG levels in the SHRs progressively increased with age and was associated with increased BP in SHRs compared with the age-matched WKY rats. In fact, MG-induced CEL and CML formation was significantly elevated in the vasculature of SHRs when compared to age-matched WKY rats (Wang et al. 2004, 2005). In this study, no observed difference in BP was seen between the SHRs and WKY rats at 5 weeks; however, this changed at 8 weeks. At this age SHRs exhibited a significant increase in systolic BP, along with a progressive increase in MG and MG-induced AGE products, CEL and CML, in both the aorta and kidney of SHRs (Wang et al. 2004, 2005). Thus, the increase of MG and MG-induced AGE formation may be a causative factor for hypertension development (Figure 1-5).

3.4.2 Diabetic complications

Not only is this reactive aldehyde connected to hypertension, MG is also linked to diabetes. In a clinically study conducted by McLellan et al (1994), they found that MG serum levels increased by 5-6 fold in type 1 diabetic patients, and by 2-3 fold in type 2 diabetic patients. Indeed, the circulating levels of MG could be due to abnormal elevations in the activities of plasma SSAO, MG-producing enzyme in protein catabolism, and in plasma AMO, MG-production enzyme in lipolysis (Ekbom 1998). Likewise, Yu et al (2003) also reported increased serum SSAO activity in patients suffering from diabetic complications, such as retinopathy and nephropathy. Since MG is an important precursor for AGE formation, plasma concentrations of imidazolone (Kilhovd et al. 2003) and argpyrimidine (Wilker et al. 2001), both MG-induced AGEs, are significantly increased in diabetics. In agreement, inhibitors of AGE formation, aminoguanidine and pyridoxamine, were demonstrated to decrease or abolish the development of retinopathy, nephropathy, and neuropathy in diabetic rats (Hammes et al. 2003; Alderson et al. 2003). Schmidt et al (1999) reported a significant increase in RAGE expression in both endothelium and VSMCs of diabetic patients. Moreover, these authors also reported that upon activation of RAGE, this induced a cascade of pro-inflammatory processes, such as the activation of NF-κB and enhanced expression of cell adhesion molecules. Thus, it was suggested by Bourajjaj et al (2003) that chronic inflammation may play a role in microvascular complications in patients with diabetes mellitus. Overall, it is highly suggestive that the maladaptive, overproduction of MG in diabetic patients may be linked to the micro- and macrovascular complications, likely due to its AGEs producing and pro-inflammatory properties.

3.4.3 Aging

Aging is a multifactorial process that affects the cell, organ, and whole body level (Desai et al. 2009). Oxidative stress is thought to play an important role in age and age-related diseases (Desai et al. 2010). Indeed the free radical theory of aging, first proposed by Denham Harman in 1956, states that throughout an organism's lifespan, the endogenous production of free radicals, inevitably produced as by-products of cellular metabolism, would ultimately react with and cause irreversible damage to cellular function and tissue constituents, eventually leading to disease and death. In fact, the main source for free radical production is from the mitochondria, specifically the electron transport chain (Harman 2001). Indeed, aged organisms show increased free radicals and oxidatively-damaged mitochondria DNA (Beckman and Ames 1998). Damaged mitochondria DNA can result in the production of dysfunctional enzymes and structurally abnormalities in the electron transport chain, which can further increase the production of ROS (Beckman and Ames 1998). Wang et al (2009) showed that MG can react with complex III, which would disrupt the flow of electrons in the electron transport chain, thus uncoupling electron flow, resulting in increased production of O2.- and ONOO- in A-10 cells. Additionally, Wang et al (2009) also showed that MG inhibited the activity of MnSOD (mitochondrial SOD), which is the first line of defence for overproduction of O2.-in the mitochondria, thus further enhancing oxidative stress in A-10 cells. Overall, being a major source of free radical and ROS production, increased MG levels can be characteristic feature of aging.

Additionally, many studies have shown that excessively high levels of glucose and caloric intake increases the status of oxidative stress, which can shorten life span (McCay et al. 1935; Weindruch and Walford 1988; Szatrowski and Nathan 1991; Simic and Bertgold 1991). As mentioned before, MG-induced AGE formation has been linked to aging (section 3.3.3) (Ando et al. 1999; Li et al. 1996). Once MG reacts with a protein, it becomes a stable complex. Because of this, the measurements of AGEs, such as CEL, are representative markers of the status oxidative stress and cumulative markers of oxidative damage to proteins in aging (Degenhardt et al. 1998; Kilhovd et al. 2003). In diabetes, most of the destructive effects of MG-induced AGE formation are mostly seen later on in age, due to the development of macro-vascular damage to the eyes, kidneys, and nerves (Sugiyama et al. 1996). Overall, the accumulation of MG and AGEs can be seen as one of causative factors in the phenomena known as aging.

4.0 Gluconeogenesis

Glucose is a major energy source for all mammalian cells; therefore, proper measures must be in place to ensure against hypoglycaemia (Pilkis and Granner 1992). Gluconeogenesis is the de novo synthesis of glucose and it occurs during periods of fasting, starvation, low-carbohydrate diets, or intense physical activity (Pilkis and Granner 1992). The rate of gluconeogenesis is determined by the unidirectional enzymes: phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase) (Marsenic 2009). These gluconeogenic enzymes are controlled at the transcriptional level by key hormones, particularly insulin, glucagon, glucorticoids, and catecholamines (Pilkis and Granner 1992). During times of starvation or intense physical activity, plasma levels of glucagon, glucocorticoids, and catecholamines will increase, leading to increased activity of G6Pase, FBPase, and PEPCK, and a coordinate decrease of glycolytic enzymatic activity (Hers and Hue 1983; Pilkis et al. 1988; Pilkis and Granner 1992) via increased intracellular levels of cAMP (Solomon et al. 1988), as well as the phosphorylation of several enzymes, and/or by changes in allosteric effectors (Pilkis et al. 1988; Hers and Van Schaftingen 1982). The opposite occurs during food consumption. By a series of mechanisms, β-cells in the pancreas will increase insulin secretion, counter-regulatory hormones decrease, resulting in suppressed glycogenolysis and gluconeogenesis and increased activity of glycolytic enzymes, including pyruvate kinase (PK), phosphofructokinase (PFK), and glucokinase (GK) (Pilkis and Granner 1992; Pilkis et al. 1988).

4.1 Gluconeogenesis and its association with T2DM

It is widely accepted that endogenous glucose production in T2DM is inappropriately increased during times of fasting and postprandial as a result of elevated gluconeogenesis (Boden et al. 2001; Magnusson et al. 1992; Gastaldelli et al. 2000; DeFronzo 1999). In fact, the rate of glucose production is increased by approximately 25-100% in patients with T2DM compared to the non-diabetic patients (Hundal et al. 2000). Endogenous glucose production occurs through two processes: glycogenolysis and gluconeogenesis. Glycogenolysis is the process that involves the breakdown of glycogen to glucose-6-phosphate and its subsequent hydrolysis by glucose-6-phosphatase to free glucose (Gerich et al. 2001), whereas, gluconeogenesis involves the de novo synthesis of glucose-6-phosphate from noncarbohydrate precursors, such as lactate, glycerol, and amino acids, where glucose-6-phosphate is subsequently hydrolysis to glucose (Gerich et al. 2001). The only organs capable of generating sufficient glucose to be released into the circulation are the liver and kidney (Gerich et al. 2001).

4.1.1 Drug therapy that targets gluconeogenesis in T2DM Metformin

Because gluconeogenesis is abnormally elevated in T2DM, it is a target of therapy for patients with diabetes. One anti-diabetic drug that targets gluconeogenesis in T2DM is metformin. Indeed, Metformin has been available for treatment of T2DM for nearly 8 years, yet it is the most widely prescribed antihyperglycemic agent (Hundal and Inzucchi 2003). It is widely held that Metformin significantly decreases plasma glucose levels in T2DM by inhibiting gluconeogenesis, and by inhibiting glucose uptake into the cell (Hundal et al. 2000; Natali and Ferrannini, 2006; Inzucchi et al. 1998; Perriello et al. 1994). Although, its precise mechanism is controversial, Metformin can decrease hepatic gluconeogenesis by inhibiting hepatic lactate uptake (Radziuk et al. 1997), reducing the intracellular hepatic ATP concentration (Argaud et al. 1993), and by inhibiting PEPCK activity (Large & Beylot, 1999). In addition, Metformin can lower plasma free fatty acids by 10-30% in the diabetic subjects (Hundal et al. 2000). Since, fatty acids are known to increase the rate of gluconeogenesis (Sindelar et al. 1997), the Metformin-induced decrease in free fatty acids may also contribute to the reduced rates of gluconeogenesis (Hundal and Inzucchi, 2003). Inhibitors of key gluconeogenic enzymes

Since the rate of gluconeogenesis is determined by G6Pase, FBPase, and PEPCK, these three enzymes form the major control points in gluconeogenesis and thus have all been targets of drug discovery efforts (Figure 1-6). However, inhibition of PEPCK and G6Pase has shown to be problematic. For example, due to PEPCK involvement in the early mitochondrial steps of gluconeogenesis, there are multiple potential side effects, such as increased mitochondrial red-ox state, inhibition of the tricarboxylic acid cycle, and a reduction in beta-oxidation of fats leading to hepatic steatosis (Burgess et al. 2004). Additionally, PEPCK inhibition does not inhibit endogenous glucose production when the substrate glycerol is in abundance (Burgess et al. 2004; Poelje et al. 2007). Inhibition of G6Pase, on the other hand, has also significant mechanistic concerns. Because G6Pase catalyzes the final step in gluconeogenesis, as well as glycogenolysis, inhibition of this enzyme represents substantial risk for the development of hypoglycaemia, which could be fatal (Poelje et al. 2007).

The development of a FBPase inhibitor, however, is thought to be a more logical target for pharmacological intervention. FBPase is the second-to-last enzyme in gluconeogenesis, meaning that it is not involved in the first step of glucose production, by the mitochondria, and not in the last step, such as the breakdown of glycogen. This theoretically reduces the risk of hypoglycaemia, lacticemia, and hyperlipidemia (Poelje et al. 2007). In fact, individuals who are genetically deficient in FBPase have near normal biochemical and clinical parameters (Gitzelmann et al. 1995). Furthermore, FBPase expression is significantly up-regulated in diabetic animal models (Kodama et al. 1994; Lamont et al. 2006). Recently, a FBPase-specific inhibitor, CS-917, completed preliminary trails suggesting clinically relevant glucose lowering was achieved in patients with T2DM without eliciting the risk of hypoglycaemia, lacticemia, and hyperlipidemia (Triscari et al. 2006; Bruce et al. 2006; Walker et al. 2006). However, a larger scale and longer-term clinical trial are still needed.

4.1.2 PGC-1α and its association with gluconeogenesis PGC-1α and its stimuli effects

The peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is a transcriptional coactivator that induces many physiological stimuli. PGC-1α is a potent stimulator of mitochondrial biogenesis and respiration (Kelly and Scrapulla 2004; Lehman et al. 2000; Lin et al. 2002; Puigserver et al. 1998). Indeed, this gives PGC-1α the ability to regulate adaptive thermogenesis in brown adipose tissue (BAT) (Puigserver et al. 1998), fiber-type switching in skeletal muscle (Lin et al. 2002), as well as stimulate β-oxidation of fatty acids and gluconeogenesis in the liver (Herzig et al. 2001; Puigserver et al. 2003; Rhee et al. 2003; Yoon et al. 2001). Up-regulation of G6Pase, FBPase, PEPCK

During the fasting state, glucagon secretion will increase, which will then increase the intracellular cAMP levels in the liver. As a result, the transcription factor, cAMP response element-binding (CREB), will become activated, thus leading to the induction of the gene expression of PGC-1α (Puigserver, 2005). Once PGC-1α is activated, it will bind to co-activators such as hepatocyte nuclear factor (HNF4α), forkhead box O1 (FOXO1), which will then lead to the induction of G6Pase, FBPase, and PEPCK gene expression (Puigserver 2005). Yoon et al (2001) showed that subjecting mice to a 24-hr fast induce a 3.7-fold increase in PGC-1α mRNA, which was reversed by refeeding. These authors also showed that by performing a time course of fasting, an increase in PGC-1α mRNA was observed after 2 hr and peaked after 5 hr of food deprivation, where PEPCK mRNA also exhibited a similar response pattern to PGC-1α. Additionally, PGC-1α markedly increased the mRNA expression of G6Pase, FBPase, and PEPCK in rat hepatocytes with an adenovirus-based vector for PGC-1α. Furthermore, when rats were injected with adenoviruses expressing PGC-1α, after 5 days, glucose output and insulin secretion was significantly elevated (Yoon et al. 2001).

During food consumption, on the other hand, gluconeogenesis will be "turned off" and glycolysis will be "turned on." Through the actions of insulin, the Akt pathway will be activated, leading to the phosphorylation of FOXO1, marking it for degradation (Puigserver 2005). Without FOXO1, PGC-1α would be unable to bind and localize to the promoter chromatin region of the gluconeogenic genes, thus significantly decreasing the transcription activity of gluconeogenic enzymes (Puigserver 2005). Meanwhile, the activities of glycolytic enzymes will increase, where the gluconeogenic enzymatic activities will decrease, leading to enhanced glucose metabolism (Puigserver 2005). Overall, insulin is the dominant regulator of gluconeogenesis. Up-regulation of ERRα

Of course, all potent regulators come with their own unique suppressors. Estrogen-related receptor-α (ERRα), is one of the first orphan nuclear receptors identified and it shares a significant sequence similarity to the estrogen receptor (ER) (Schreiber et al. 2003). ERRα, along with ERRβ and ERRγ, recognize and bind to similar DNA sequences recognized by ERs, however the in vivo function of ERRα is still unclear (Schreiber et al. 2003). PGC-1α and ERRα are both predominately expressed in organs with high metabolic needs such as the skeletal muscle and kidneys (Ichida et al. 2002). Indeed, when Ichida et al (2002) showed that after starving mice overnight, both PGC-1α and ERRα were significantly up-regulated at the transcriptional level. In fact, when PGC-1α is induced, it regulates the expression of ERRα mRNA, as well as its transcriptional activity (Schreiber et al. 2003), where in turn, ERRα can significantly repress PGC-1α transcriptional activity (Ichida et al. 2002; Herzog et al. 2006). Thus, ERRα has opposing effects on genes important for gluconeogenesis. PGC-1α and its induction in ROS-detoxifying enzymes

Additionally, PGC-1α is also a broad and powerful regulator of ROS metabolism (St-Pierre et al. 2006). This is due to PGC-1α ability to be a potent stimulator of mitochondrial biogenesis, which would consequently introduce the production of ROS. Mitochondrial metabolism is responsible for the majority of ROS production in cells (Balaban et al. 2005). This occurs when unpaired elections escape from the electron transport chain and react with molecular oxygen, generating O2.-, and thus ONOO- (Brown & Borutaite, 2001).

ROS-detoxifying enzymes are the first line of defense to combat the deleterious effects of excess ROS production. These anti-oxidant enzymes include, but are not limited to, SOD1 and manganese SOD2, catalase, and GSH-Px (St-Pierre et al. 2006). In fact, Bruce Spiegelman and associates (2006) showed that PGC-1α can stimulate the mitochondrial electron transport while suppress ROS levels, which is accomplished by increasing the expression and activity of SOD1 and 2, catalase, and GSH-Px (Figure 1-7). This mechanism thus allows tissues, such as brown fat and skeletal muscle, to increase mitochondrial metabolism without causing self-inflicted oxidative damage (St-Pierre et al. 2006). The authors also reported that PGC-1α null mice displayed a blunted induction of the ROS defense system and were more sensitive to oxidative stress in comparison to their wild type counterparts. For example, Spiegelman and associates (2006) showed that approximately half of the PGC-1α null mice died after exposure to 1.5 mM H2O2 while only 25% of the wild type counterparts died. Furthermore, under 1.5 mM H2O2, more than 80% deaths occurs in cells lacking both PGC-1α and -β (St-Pierre et al. 2006). Thus, PGC-1α may serve as an adaptive regulator of ROS production, ensuring balance between metabolic requirements of cell and its cytotoxic protection. PGC-1α and its interaction with NO and CO

There are many upstream effectors that can induce PGC-1α expression and activity. For instance, in BAT and liver tissues, the β-adrenergic/cAMP pathways activates PGC1A gene transcription and calcineurin A and calcium/calmodulin-dependent protein kinase (CaMK) activates PGC-1α expression in striated muscles (Finck and Kelly 2006). Recently, NO, a gasotransmitter originally identified as a vasodilator, was shown to activate mitochondrial biogenesis through the induction of PGC-1α (Figure 1-8) (Nisoli et al. 2003). Nisoli et al (2003) found that overexpression of NO, cGMP, or eNOS significantly increased mitochondrial numbers in cells as diverse as brown adipocytes and 3T3-L1 (mouse white fat cell line), U937 (human monocytic cell line), and HeLa (human cervical cancer cell line) cells. Furthermore, both male and female eNOS null mice exhibited decreased numbers of mitochondria in brain, liver, and heart tissues, which resulted in decreased energy metabolism and weight gain (Nisoli et al. 2003).

Additionally, another gasotransmitter, CO, was also shown to also be involved in adaptive oxidative metabolism by optimizing mitochondrial biogenesis (Suliman et al. 2007). Suliman and associates showed that in the mouse heart and in isolated cardiomyocytes, that by activating both guanylate cyclase, as well as pro-survival kinase Akt/PKB, CO also induced the expression and activity of PGC-1α. It has yet to be determined whether H2S could also induce the expression of PGC-1α. PGC-1α and its association with diabetes mellitus

Both animal and human studies have shown that altered PGC-1α signaling could lead to glucose intolerance, insulin resistance, and diabetes (Yoon et al. 2001; Koo et al. 2004; Andrulionyte et al. 2004; Ek et al. 2001; Hara et al. 2002; Vimaleswaran et al. 2005; Oberkofler et al. 2004). PGC-1α activity is abnormally elevated in diabetic liver in the fasted state (Finck and Kelly 2006), which could be a main contributing factor for hyperglycemia. Additionally, PGC-1α may promote insulin resistance by inducing TRB-3, an inhibitor of Akt signaling, thus interfering with insulin signaling (Koo et al. 2004). In fact, FBPase, a downstream target of PGC-1α, was up-regulated 5-fold in pancreatic islets from diabetes-susceptible obese BTBR mice compared with the diabetes-resistant C57BL/6 mice (Lan et al. 2003). Additionally, Kebede et al (2008) showed that up-regulation of FBPase can have detrimental effects to β-cells, because it can decrease cell proliferation rate, as well as impair insulin secretion by depressing glucose-induced insulin secretion. Thus, FBPase overexpression in β-cells results could result in reduced glycolytic flux and energy production (Kebede et al. 2008). However, the precise mechanism by which cross-talk occurs between insulin signaling and PGC-1α activity, in the diabetic state, is currently unknown and is an active field in diabetic research (Finck and Kelly 2006).

4.3 Renal gluconeogenesis

The kidney plays a vital role in BP regulation (Tomaschitz et al. 2010), but its role in glucose metabolism is often ignored (Gerich and Meyer 2001). Until recently, it was believed that the liver was solely responsible for gluconeogenesis, and that renal gluconeogenesis became significant only during prolonged fasting or acidosis (Gerich 2000; Gerich et al. 2001; Roden and Bernroider 2003). However, it is now recognized that the kidney has a significant role in glucose homeostasis via gluconeogenesis and reabsorption of filtered glucose (Meyer et al. 2002a; Gerich et al. 2001; Meyer et al. 2004). Indeed, renal gluconeogenesis has been estimated to account for ~20 ± 2% of total glucose release (Gerich and Meyer 2001). However, in relation to T2DM, renal release of glucose is significantly elevated in the fasting state. Meyer et al (1998) showed that the absolute increase in renal glucose release is comparable to that of the liver in magnitude (2.60 and 2.21 µmol/(kg min) for liver and kidneys, respectively). In fact, the relative increase in renal gluconeogenesis is substantially greater than the increase in hepatic gluconeogenesis (300 vs. 30%) (Meyer et al. 1998).

The proximal tubule, located within the renal cortex, is the only segment of the nephron capable of gluconeogenesis, because this is the precise location of the key gluconeogenic enzymes such as G6Pase, FBPase, and PEPCK (Gerich 2000; Guder and Ross 1984; Schoolwerth et al. 1988). Indeed, these three gluconeogenic enzymes are active along the entire length of the proximal tubule (Conjard et al. 2001).

5.0 Rationale and hypothesis

Recent studies have concluded that MG regulates signal transduction systems, balances redox reactions, as well as influence cell survival. However, abnormally high levels of MG and MG-induced production of ROS and AGEs are implicated in the alternation of vascular reactivity, wall inflammation, oxidative stress, and endothelial dysfunction. H2S, on other hand, can induce reconditioning and cardiac protective effects. For example, H2S is a scavenger for ROS and RNS and can indirectly increase GSH levels, thus combating oxidative stress, as well as reducing inflammation and promoting cellular apoptosis. However, overproduction of H2S has been linked to the pathogenesis of septic shock and diabetes.

With this in mind, the cell must have a highly sophisticated regulation mechanism(s) in place in order to tightly control these potent endogenous and influential molecules. Thus, it is logical to assume that a possible link exists between H2S and MG. Since MG and H2S are both involved in opposing pathways (pro-oxidant vs. anti-oxidant, induce proliferation vs. apoptosis, pro-inflammatory vs. anti-inflammatory), it is possible a negative correlation exists between MG-induced responses and H2S-induced effects. Elucidation of a possible relationship between MG and H2S in physiological and pathophysiological conditions could lead to more elaborate and effective therapeutic treatments to combat the complex array of oxidative stress and its implications.

A crosstalk phenomenon may occur between MG and H2S, such as the down-regulation of endogenous synthesis of the opposing molecule, as well as the attenuation of its downstream effect, possibly to maintain balance in the cell. If crosstalk does occur between MG and H2S, it may play an important role in the overall picture of cellular physiology, were a disruption of this scale may lead to different pathophysiological conditions. Therefore, my hypothesis is that a physiological balance between MG and H2S plays an important role in the regulation of glucose metabolism; an imbalance in this relationship may be one contributing factor in the development of some metabolic disorders (Figure 1-9)

Figure 1-9: Hypothesis: physiological balance between MG and H2S is needed to maintain normal glucose metabolism and cellular function.

6. 0 Objectives and experimental approaches

This thesis is mainly focusing on the possible connection between MG and H2S, where not much is known in this field. Therefore, this project has been divided into 3 consecutive studies.

6.1 Study 1: Interactions of methylglyoxal and hydrogen sulfide in rat vascular smooth muscle cells

MG and H2S are both produced in vascular tissues, we first need to determine if an interaction, either direct or indirect occurs, and what are the physiological outcomes (Figure 1-10). Thus, in order to accurately study the in vivo and in vitro interaction of MG and H2S we needed to:

Determine if a chemical-to-chemical interaction can occur between MG and H2S in a cell-free medium.

Investigate if administration of MG and H2S will decrease the endogenous level of one another in A-10 cells.

Study the oxidative stress of A-10 cells when exposed to exogenous levels of MG, H2S, or both by using the DCF assay. Furthermore, it would be essential to analyze any changes within the endogenous levels of L-cysteine, homocysteine, and GSH.

Analyze whether or not MG can affect the expression levels and activity of the dominant H2S-producing enzyme in the cardiovascular, CSE, since MG is known to reactive and modify proteins.

This study would provide us with the novel insight if cross-talk, does in fact, occur between a gasotransmitter and a reactive glucose metabolic can take place in rat thoracic aortic VSMCs (A-10 cells), whether it be indirect and/or direct. These discoveries may help unveil complex pathologic mechanisms of various diseases such as hypertension and other forms of insulin resistance syndrome with altered cysteine/homocysteine metabolism.

Figure 1-10: Schematic diagram for the layout of Study 1 in A-10 cells.

6.2 Study 2: Increased methylglyoxal formation in kidneys of male mice with deletion of cystathionine γ-lyase

After observing that H2S has cytoprotective properties against MG, due to its MG scavenging abilities, abolishing MG-induced ROS production, and upregulating GSH expression levels, we sought to determine whether MG levels and gluconeogenic enzymes are altered in kidneys of 6-22 week-old CSE-/- male mice. These genetically knockdown mice were generated by our lab and collaborators, where both male and female CSE-/- mice developed hypertension (Yang et al., 2008). The kidney was the tissue of choice in this study, because these organs have high metabolic rates of MG, and thus would provide us an accurate assessment of any alternation in MG formation pathway. Thus, to investigate if a physiological balance occurs between MG and H2S, we plan to analysis age-related changes in MG levels, gluconeogenic enzymes, and transcription factors in the kidneys of CSE-/- male mice, ages 6-22 weeks old (Figure 1-11), with the intention of determining:

Altered plasma glucose and MG levels in 6-22 week-old CSE-/- and CSE+/+ mice.

Age-influenced changes in the MG levels, along with the MG precursors, DHAP and GA3P, in renal tissues of CSE-/- and CSE+/+ mice.

Age-related alternations of the enzymatic activities of FBPase, which catalyzes the conversion of fructose-1,6-bisphosphate (F-1,6-P) to fructose-6-phosphate (F-6-P), and the counter glycolysis enzyme, PFK, which catalyzes the conversion of F-6-P to F-1,6-P of the kidney of CSE-/- and CSE+/+ mice. We also plan to analysis the F-1,6-P and F-6-P levels in the renal tissues of CSE-/- and CSE+/+ mice.

Alternated mRNA levels of the main rate-limiting gluconeogenic enzymes, FBPase-1,-2 and PEPCK, along with the mRNA levels of the gluconeogenic regulators, PGC-1α and ERRα, in the CSE-/- and CSE+/+ mice.

Age-related changes in the oxidative status of the kidney of CSE-/- mice by measuring tissue GSH levels.

Performing a gluconeogenic study in mice with the deletion of CSE, and thus lower levels of vascular H2S, would provide us with the information needed to determine if the endogenous production of MG can be mediated by H2S, likely by influencing the rate of gluconeogenesis. Additionally, in regards to the drug activity of Metformin, possible alternations of FBPase in the presence of decreased H2S, could provide novel insight to one researching FBPase inhibitors to treat hyperglycemia in diabetic patients. Figure 1-11 summarizes our experimental objectives.

Figure 1-11: Schematic diagram for the layout of Study 2 in the kidney of CSE-/- male mice