Homocysteine and reactive oxygen species in metabolic syndrome, type 2 diabetes mellitus, and atheroscleropathy: The pleiotropic effects of folate supplementation

ROS consist primarily of the oxygen free radicals (superoxide [O ₂ ^•- ], hydroxyl radical [-OH ^•- ], peroxynitrite [ONOO ^•- ]), the potent oxidizing agents of the non-radical family (peroxide [H ₂ O ₂ ] and hypochlorous acid [HCLO]), and the organic analogues, which include reactive nitrogen species: specifically, peroxynitrite [ONOO ^•- ] (table 5).

It has been known for some time that ROS are detrimental and toxic to cells and tissues as a result of injury to lipids, nucleic acids, and proteins: (A). Lipid peroxidation of membranes (loss of membrane function and increased permeability) and generation of lipid autoperoxidation reactions. (B). DNA damage leading to mutation and death. (C). Cross linking or vulcanization of sulfhydryl rich proteins (leading to stiff aged proteins specifically collagen of the extracellular matrix) [32].

Both intracellular and extracellular antioxidants play important roles in neutralizing these toxic oxygen molecules. The endogenous antioxidant enzyme family consists primarily of superoxide dismutase (SOD), catalase, glutathione peroxidase and the tri-peptide (gamma-L-glutamyl-L-cysteintyl-glycine) glutathione or (GSH), and free circulating thiols containing the sulfhydryl group (-SH) (table 6). These antioxidants are found to be systemically depleted in the clinical setting of T2DM and evidence is present for their depletion in atheromatous lesions [31, 32].

There are multiple metabolic toxicities associated with MS, PD, T2DM, and their companion: accelerated atherosclerosis (atheroscleropathy). These multiple metabolic toxicities can be conveniently grouped in an acronym termed the A-FLIGHT toxicities (table 4). Each of these toxicity substrates result in the excessive production of ROS responsible for the damaging effects of oxidative – redox stress.

Not only are ROS involved in the development of autoimmune type 1 diabetes mellitus and T2DM but also play an important role in the long-term development of the associated complications: atheroscleropathy, diabetic cardiomyopathy, intimopathy, nephropathy, neuropathy, endotheliopathy, and retinopathy.

There are multiple metabolic toxicities associated with MS, PD, and overt T2DM, which are associated with the production of ROS. These toxic substrates and their associated ROS have been conveniently grouped together to form the acronym: A-FLIGHT toxicities (table 4).

The general population of patients with MS, IR, PD, and overt T2DM will, in allprobability, have a similar frequency of the common polymorphism affecting the folate-dependent, thermolabile gene coding for the 5, 10-methylene tetrahydrofolate reductase MTHFR) ((C677T, Ala → Val) is associated with a decreased activity of the enzyme with subsequent mild to moderate HHcy, which occurs in 10–15% of the general population [33‐35].

This gene polymorphism is especially important in those individuals with a decrease in dietary folate. In these patients mild to moderate HHcy can be improved with dietary and folate supplementation, which improves endothelial-dependent endothelial cell dysfunction [36]. In the general population, mild to moderate HHcy occurs in approximately 5–7% [9].

HHcy is not usually present as a direct result of MS, PD, and T2DM unless there is an associated development of impaired renal function. As nephropathy develops, there is an associated elevation of total Hcy associated with a decline in glomerular filtration rate [37].

In the early latent Stage I and transitional Stage II of T2DM (table 7) homocysteine may actually be lower than expected. Rosolova H et al. have found an unexpected inverse relationship between IR and serum Hcy in a population of healthy subjects with insulin resistance. They have attributed this finding to the associated increase in the glomerular hyperfiltration [38].

IR and PD (Stages I, II, and early Stage III) are associated for a period of time with hyperinsulinemia, hyperproinsulinemia, and hyperamylinemia and glomerular hyperfiltration and hyperperfusion. Recently, the nitric oxide system has been shown to be involved in glomerular hyperfiltration in Japanese normo- and micro-albuminuric patients with T2DM and early stages of diabetic nephropathy [39]. Hiragushi K et al. were able to demonstrate an increase staining of eNOS enzyme (in glomerular endothelial cells) in microalbuminuric T2DM patients in comparison to non-diabetic control subjects.

This adaptive early response results in glomerular enlargement and an increase in the glomerular filtration rate [40]. Additionally, Apakkan Aksun S et al. found that serum and urine nitric oxide levels were elevated, as well as, plasma malanodialdehyde (MDA), which points to the importance of oxidative stress in patients with T2DM. These authors feel that the high nitric oxide levels may lead to hyperfiltration and hyperperfusion, which in turn leads to an increase in urinary albumin excretion and thus causes progression of nephropathy in early T2DM [41].

Over time (Stages III-V, table 7) there is an increase in glucotoxicity (initially postprandial Stage III and fasting in Stage IV), as well as, an increase in redox and oxidative stress to the glomerular endothelium. This damage is associated with lipid peroxides, free fatty acids, and activation of Ang II as a result of hyperinsulinemia, hyperproinsulinemia, hyperamylinemia and the totality of reactive oxygen species associated with the A-FLIGHT toxicities.

The activation of Ang II would be associated with the activation of the membranous NAD(P)H oxidase enzyme and generation of superoxide with its effect on endothelial cell dysfunction and uncoupling of the eNOS reaction. Over time, this would result in decreases in eNO and loss of a natural occurring local antioxidant network within the glomerular endothelial microenvironment.

Glucotoxicity is associated with advanced glycosylation endproducts (AGEs) and their receptor (RAGE), activation of aldose reductase of the polyol-sorbitol pathway, activation of protein kinase C (PKC) isoforms, and transforming growth factor beta (TGFbeta). Once these glucotoxic mechanisms are in play and overt diabetic nephropathy with associated glomerulosclerosis and remodeling has developed, there will be a progressive decrease in glomerular filtration. These changes will result in Hcy elevation [37].

Thus, HHcy plays an extremely important role for additional oxidative-redox stress regarding the accelerated cardiovascular disease observed in diabetic patients on dialysis [42].

HHcy is thought to induce an oxidative inactivation of endothelial nitric oxide, in part by inhibiting or consuming the expression of cellular glutathione peroxidase (GPx). In heterozygous cystathionine beta-synthase deficient -/+ mice, Weiss et al. were able to restore endothelial cell function by increasing cellular thiols and reduced glutathione pools and increasing GPx activity with restoration of the endothelial function [43].

The ensuing cellular redox stress is magnified and total Hcy consumes NO by the indirect process of superoxide (O₂ ^-) converting NO to toxic peroxynitrite (ONOO^-).

In addition to ONOO^- formation, NO in conjunction with thiols and oxygen radicals generate nitrotyrosine and nitroarginine. Nitroarginine, in turn, competes for the substrate L-arginine of the eNOS reaction in a feedback mechanism, limiting further NO generation [44‐46]. As a result, there is endothelial cell dysfunction, endothelial cell toxicity and endothelial cell loss-apoptosis, increased ROS, increased ONOO^- and decreased NO associated with HHcy [47]. HHcy is multiplicative in nature and even though its effects may occur later in T2DM than the other associated A-FLIGHT substrate toxicities, it has a devastating – synergistic effect on endothelial cell function. Presently, we know there are other multiple metabolic toxicities (table 4) operating within the renal glomerulus producing microalbuminuria (reflecting endothelial cell dysfunction and damage) at a stage prior to the declining glomerular filtration rate responsible for HHcy (figure 2).

A recent clinical study by Maejima et al. [48] revealed significant elevated levels of ONOO^- peroxynitrite (by Griess method) in 129 T2DM patients as compared to 76 non-diabetic controls. While there was no difference in basal plasma NO(2)(-) levels between the T2DM and control patients, ONOO^- levels were related to the presence of hypertension and advanced microvascular complications. In addition, ONOO^- correlated positively with elevations in AGEs and serum lipid peroxide. These data support the hypothesis that decreased endothelium-dependent vasodilatation in diabetic subjects is associated with the impaired action of NO secondary to its consumption (quenching) from redox stress rather than decreased NO production from vascular endothelium. Clinically, abnormal NO metabolism is related to advanced diabetic microvascular complications.

Zhang et al. [49] were able to demonstrate that increased concentrations of Hcy resulted in a decreased NO response to bradykinin and L-arginine. Hcy stimulated the formation of superoxide anions and peroxynitrite with increased levels of nitrotyrosine. The addition of 5-methyltetrahydrofolate (folic acid) restored NO responses to bradykinin and L-arginine agonists. In addition, scavengers of peroxynitrite and SOD mimetics reversed the Hcy-induced suppression of NO production by endothelial cells. Concentrations of Hcy (greater than 20 micromolar concentration) produced a significant indirect suppression of eNOS activity without any discernible effects on its expression.

Li et al. just published an article showing an unexpected effect of Hcy-induced oxidative stress resulting in an increase of 3-hydroxy-3-methylglutaryl coenzyme A reductase in vascular endothelial cells, as well as decreasing endothelial NO. They were also able to demonstrate that "statins" increased NO as well as decreasing cellular cholesterol [50].

Stuhlinger et al were able to demonstrate that Hcy impairs the nitric oxide synthase pathway. Homocysteine inhibits dimethylarginine dimethylaminohydrolase (DDAH), which is responsible for degrading ADMA. This effect of Hcy causes the endogenous inhibitor of nitric oxide synthase, ADMA, to accumulate and compete with L-arginine for nitric oxide production. This effect helps to explain the deleterious effect of Hcy on the endothelial cells ability to promote vasodilatation and associated endothelial cell dysfunction with decreased NO synthesis [51].

Previous publications have emphasized the importance of the eNOS enzyme and the important role of the Glu²⁹⁸ → Asp gene polymorphism [32, 52]. Noiri and colleagues have recently demonstrated that the gene polymorphism (Glu²⁹⁸ → Asp) affecting the eNOS enzyme is statistically and significantly associated with diabetic nephropathy in T2DM patients [53]. This finding points to the important role of the eNOS enzyme and eNO in controlling oxidative – redox stress and its relation to the development of diabetic nephropathy.

This same gene polymorphism (Glu298 → Asp) of the eNOS enzyme (eNOS 894TT genotype) has also been recently demonstrated to be a risk factor for HHcy. These findings by Brown et al. indicate that the NOS3 894TT genotype (eNOS Glu298 → Asp gene polymorphism) is a risk factor for HHcy in healthy nonsmoking adults with low serum folate and supports the hypothesis that nitric oxide modulates Hcy through an effect on folate catabolism [54].

The above discussion also applies to the accelerated atherosclerosis (atheroscleropathy) and intimal redox stress and remodeling (intimopathy) associated with MS, PD, and overt T2DM [32].

Insulin resistance is central to the development of CHD and the accelerated atheroscleropathy associated with MS, PD, and overt T2DM. This being said, the ultimate manifestation of insulin resistance is T2DM [32] (figure 3).

Atherosclerosis, MS, PD, and T2DM present a pleiotropic mechanistic progression, in which, inflammation and upstream oxidative-redox stress and ROS appear to share a common – fertile soil background, and patients with diabetes experience both a preexisting and parallel-accelerated atherosclerosis, we have termed atheroscleropathy [32, 52, 55] (figure 4).

In regards to silent coronary artery disease (CAD), Gazzaruso C et al. were able to confirm that microalbuminuria and smoking may predict silent CAD in people with diabetes and additionally they were able to demonstrate an original finding of an independent association between HHcy and silent CAD [56].

In the Framingham offspring study [57], those with hyperinsulinemia had significantly higher Hcy levels than those without, and subjects with more than two MS phenotypes (hypertension, impaired glucose tolerance, or a central MS [consisting of two or more of the following: hyperinsulinemia, obesity, or dyslipidemia]) had significantly higher Hcy levels compared to those with one or no MS phenotype.

Passaro and colleagues have been able to demonstrate in T2DM patients that Hcy levels are influenced by both the duration of the disease and metabolic control. Hcy levels improve with improvement of glycemic control of HbA1c. Hcy remained strongly associated to the presence of CHD independent from age, sex, body mass index, smoking status, hypertension and lipid patterns. Additionally, they conclude that Hcy can be seen as a major risk factor in T2DM patients with CHD [58, 59].

The initial vascular lesions described by McCully in 1969 [2] described an arteriosclerotic fibrotic change within the intima, which is in contrast to the intimal fibrous-lipid laden lesions associated with atherosclerosis and the vulnerable thin-cap fibroatheroma prone to rupture.

Burke and colleagues have been the first to describe the plaque morphology and correlate these findings to total plasma homocysteine [60]. Their recent publication demonstrated that Hcy is elevated in sudden unexpected death in men as a result of severe coronary artery disease without acute or organized coronary thrombosis. Additionally, they were able to demonstrate that the above association seemed to be increased if there existed concomitant diabetes mellitus. HHcy was associated with an increase in fibrous plaques and a relative decrease in thin-cap atheromas. Their findings suggest a different mechanism of atherogenesis from that of hypercholesterolemia. Additionally, efforts to reduce Hcy could be complementary to lipid lowering therapy because plaque fibrosis may occur via a distinct mechanism. They suggest that one such mechanism could be endothelial dysfunction, which certainly points to the importance of the "Folate Shuttle phenomenon" within the endothelial cell and a relative endogenous endothelial folate deficiency within the endothelial microenvironment milieu.

These morphological findings suggest the need for; and importance of; global risk reduction of the entire spectrum of elevated substrates within the A-FLIGHT multiple metabolic toxicities.

Peroxisome proliferator activated receptors (PPARs) are transcription factors belonging to the nuclear receptor super-family and the multiple roles of their effects are rapidly unfolding.

Clinically there are two classes of drugs currently in use: fibrates binding to PPAR alpha (gemfibrozil and fenofibrate) and thiazolidinediones (TZDs) binding primarily to PPAR gamma (rosiglitazone and pioglitazone). PPAR alpha agonists are responsible primarily for their hypolipidemic effect, while PPAR gamma agonists are antidiabetic agents – insulin sensitizers (enhancing insulin – mediated glucose uptake and lowering of HbA_1C), important in the regulation of the lipid metabolism and adipogenesis, and additionally expressed in the vasculature (endothelial cells, smooth muscle cells, and monocyte – macrophage cells). In vitro and clinical data show that TZDs can decrease thrombotic, inflammatory, and oxidative changes that contribute to endothelial dysfunction and their future vaculoprotective roles (table 8) in attenuating the progressive role of non-diabetic atherosclerosis and the accelerated atherosclerosis in MS, PD, and overt T2DM [61‐67].

Current available information suggests that PPAR-gamma ligands may prevent the progression of insulin resistance (preserving islet Beta cell function) to diabetes and endothelial dysfunction to atherosclerosis [68].

Recently, Fonseca V. et al. were able to demonstrate that the PPAR agonist troglitazone had a significant Hcy lowering effect in both the lean and fatty Zucker rat model of insulin resistance. This decline in Hcy was associated with significant increases in hepatic concentrations of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) plus SAH. Additionally, there was a significant decline in the SAM/SAH ratio. The hepatic CBS was significantly higher in the troglitazone treated group as compared to the control group. The authors conclude that the Hcy lowering effect of troglitazone may be due to a shift in the concentrations of Hcy from the blood to the liver, as well as, a possible upregulation of hepatic CBS [69].

Tao et al. just published a timely article that ties together quite nicely the detrimental role of oxidative-redox stress and ROS and the antioxidant, antinitrative, and vasculoprotective role of PPARs. They were able to demonstrate (in hypercholesterolemic rabbits) that treatment with the PPAR gamma agonist rosiglitazone enhanced PPARgamma expression, improved endothelium-dependent vasodilatation, preserved the phosphorylation of vasodilator-stimulated phosphoprotein (P-VASP), suppressed gp91(phox) of the NAD(P)H Oxidase enzyme and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation [70]. Even though Hcy levels were not part of this publication, a reasonable assumption would be that rosiglitazone could also lower Hcy levels.

Recently, we have been able to demonstrate an important finding regarding the interactions of Hcy with PPAR alpha and gamma receptors. In addition to demonstrating that HHcy decreases bioavailability of eNO, generates nitrotyrosine, and activates latent matrix metalloproteinase (which instigates endomyocardial endothelial fibrosis and dysfunction) in vivo, we have been able to demonstrate that Hcy acts in a competitive manner with the PPAR receptor (in endomyocardial endothelial cells of mouse myocardium) in vitro. Specifically, Hcy interacts with the fibric acid PPAR alpha agonist ciprofibrate and the PPAR gamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ₂) [71‐74].

This may have definite clinical applications regarding the actions of the PPAR agonist family of fibric acid, as well as the PPAR agonists: TDZs (rosiglitazone and pioglitazone currently in clinical use). If Hcy is elevated there may exist a competitive inhibition of the agonist effects of both of these clinically useful medications (figure 5).

These findings of the potential interaction of Hcy with the PPAR ligands may interfere with the positive role of glucose -lipid regulation and anti-inflammatory antiatherosclerotic actions of the fibric acid derivatives and thiazolidinediones.

Flynn MA et al. have discussed atherogenesis and the Homocysteine-Folate-Cobalamin (HFC) Triad and it seems pertinent to expand each of their respective roles in this discussion [75].

The toxic role of Hcy on the vasculature has been extensively discussed previously, however, the important role of folic acid and cobalamin (vitamin B₁₂) needs further development.