Diabetes mellitus is the most prevalent metabolic disorder in the United States, with costs for treatment and care of diagnosed patients running over $174 billion. The pathological pathway of the disease revolves around inappropriate glucose metabolism, which occurs as a result of absolute deficiency of insulin secretion, reduction in the biological effectiveness of insulin, or a combination of the two. Dysfunctional insulin, in turn, leads to hyperglycemic conditions. 80-90% of cases presented in the United States are diagnosed with Type II diabetes mellitus, which is an increased risk factor for a number of deadly conditions: Read More>> 

By Nathan S. Bryan, Ph.D. 

Diabetes mellitus is the most prevalent metabolic disorder in the United States, with costs for treatment and care of diagnosed patients running over $174 billion. The pathological pathway of the disease revolves around inappropriate glucose metabolism, which occurs as a result of absolute deficiency of insulin secretion, reduction in the biological effectiveness of insulin, or a combination of the two. Dysfunctional insulin, in turn, leads to hyperglycemic conditions. 80-90% of cases presented in the United States are diagnosed with Type II diabetes mellitus, which is an increased risk factor for a number of deadly conditions: heart disease and stroke, high blood pressure, blindness, kidney disease, nervous system disease, complications leading to amputation, and complications of pregnancy and surgery, to name a few. Arguably not coincidental, all of the conditions mentioned above also are associated with insufficient nitric oxide (NO) production [1]. Furthermore, endothelial dysfunction, which leads to reduced NO levels and bioavailability, plays a major role in the pathogenesis of diabetic vascular disease, and thus could provide the link between metabolic disorders (diabetes) and cardiovascular disease. Current anti-diabetic drugs are effective in managing blood glucose levels, but have highly adverse side effects that result in the increased incidence of CV complications, including heart attack and stroke. A NO based therapeutic regime, administered in conjunction with anti-diabetic rugs, could result in an improved prognosis; managed glucose levels and amelioration of the cardiovascular complications.

The Diabetes Burden:

About 7.8 percent of the United States population suffers from diabetes, which, according to the 2007 National Diabetes Statistics of the National Institutes of Health (NIH), is about 23.6 million people. These 23.6 million people have a risk of death twice that of their age match counterparts, which can be primarily attributed to their diabetes acting as a major independent risk factor for coronary artery disease [4,5]. In 2004, heart disease was the cause of death on 68% of diabetes-related death certificates in people 65 years of age or older.  Adults with diabetes are 2-4 times more prone to heart-disease related deaths than their non-diabetic counterparts.

Diabetes also results in a 2-4 fold increase in the risk of stroke, a marked increase in blood pressure, and contributes to about 60% of non-traumatic lower limb amputations. All these pathologies mount to around $116 billion in direct medical costs and an additional $58 billion in indirect costs (disability, work loss, premature mortality). After a numerical adjustment for age and sex differences, it is estimated that average medical expenditures amongst people with diabetes is around 2.3 times higher than those without. As aforementioned, the link between endothelial dysfunction and vascular disease in the diabetic provides a salient strategy, via nitric oxide, in managing the patient’s quality of life. Eventually, this management could even lead to a reduction in the overall cost of diabetes on the entire health care system.

The nitric oxide metabolism and implications on pathogenesis of diabetes mellitus:

A healthy, functional endothelium synthesizes and secretes a number of factors and signaling molecules that are crucial in CV maintenance. NO, generated by the constitutive enzyme, Nitric Oxide Synthase (NOS), is one such factor that is essential for normal physiological regulation of blood flow and nutrient delivery to tissues; NO also doubles as an anti-leukocyte and anti-platelet factor 7]. NO is such an important signaling molecule that the absence of it, or a deficiency in the production of it, is considered to be one of the earliest indicators of disease. A loss of function of NOS is shown to have a direct correlation with major CV risk factors, such as hyperlipidemia, diabetes, hypertension, smoking and severity of atherosclerosis. NOS activity might also provide a degree of predictive potential for atherosclerotic disease progression [8-11]. Interestingly enough, in vitro animal models of diabetes mellitus have shown a tendency towards endothelial dysfunction [12-15, 79]. Furthermore, isolated rat aorta from diabetic rats show a deficiency in endothelial NO release that can only be partially rescued by supplementation by the NO precursor, L-Arginine [12-14, 16]. Most commercial supplements target NO restoration through this L-Arginine supplementation channel, and thus aren’t very effective in restoring NO levels.  This also suggests that decreased NO levels aren’t primarily based on reduced L-Arginine levels, but rather, are rooted at another source. Such a source might be the onset of endothelial dysfunction, which is extremely common in patients of diabetes mellitus [17,18]. These patients normally test low for NO, and also exhibit cardiovascular complications, which aren’t necessarily mutually exclusive. There are a variety of mechanisms which are affected by diabetes mellitus that might lead to reduced eNOS function, and in turn, reduced NO levels.

Type II diabetes mellitus, by definition, causes a spike in blood glucose, insulin and cytokine levels. In vitro, every single one of these factors has been shown to hinder eNOS activity [14,15]. Additionally, diabetes also causes an increase in advanced glycosylation products in the blood serum; these products act as quenching agents for NO, further exacerbating endothelium dependent functionality [19-21, 80]. In vitro, isolated femoral, renal and renal vessels also show decreased vasodilation when exposed to glycosylated hemoglobin [20].  One more mechanism that could provide the link between diabetes and endothelial dysfunction is mediated by vessel wall oxidant production. Under diabetic conditions, smooth muscle cells and the endothelium tend to produce reactive oxygen species, which have been shown to attenuate eNOS and endothelial functionality [22]. Furthermore, recent studies have shown that high glucose serum levels and increased glycosylation products, such as methylglyoxal (MG), increase protein expression of arginase [81].

The additive effect of these factors on the eNOS system in the diabetic population leads to a significant decrease in vascular NO production, which in turn reduces circulation to a variety of organs. Reduced circulation to organs can present a number of complications, especially in the cardiovascular system. This reduced blood flow to the heart could result in the high prevalence of CVD in diabetics. Therefore, replenishing NO could greatly reduce the burden of CVD in the diabetes realm, and in turn improve disease prognosis and reduce the financial burden of CVD in diabetic and non-diabetic populations.  

To date, trials revolving around L-Arginine supplementation and antioxidants have consistently failed, with the only exception being the success of a D-Arginine based trial whose results were specific to limiting arginase activity [81]. Organic nitrate, inhalative NO therapy and downstream phosphodiasterase inhibitors constitute a majority of the therapeutics on the market today, but supplements that employ L-Citrulline (a feedback activator of L-Arginine) and ingredients proven to bolster eNOS activity have shown to be most effective in treating endothelial dysfunction. Such therapies, if normalized in the diabetic population, could greatly reduce the CVD implications of diabetes and improve vascular health.

Insulin and NO signaling: 

Insulin, in conjunction with endothelial NO, acts as a vasodialator [23]. In fact, the major role of insulin as a cardio-protective agent results from the phosphatidylinositol 3′-kinase-protein kinase B-endothelial NOS (PI3K-AkteNOS)-dependent signalling mechanism; this along with its metabolic regulation renders insulin as an important organ protector. Furthermore, the aforementioned PI3K kinase pathway acts to phosphorylate eNOS and increase NO production [24]. This pathway is crucial, in fact, as impariment in PI3K signaling can be dually interpreted as insulin resistance at the molecular level [25].

The apparent parallelism between metabolic insulin signaling in glucose uptake and vasodilation provides important insight into the link between diabetes and endothelial dysfunciton. In fact, it has been proven that GLUT4 receptor translocation impairment in adipocytes and muscle cells conincides with eNOS impairment in the endothelium, when the PI3K pathway is impaired due to insulin resistance [27, 82]. As expected by the argument presented above, both point mutations in human subjects and homozygous null mice tests of the insulin receptor substrate 1 gene (IRS-1) have been shown to cause impaired endothilium derived vasodialation and insulin resistance; alternatively, these case studies have also cast light on the genetic link for endothelial dysfunction [27,28]. 

In more recent studies, another pathway has been linked into the PI3KAKT mechanism. Mammalian target of rapamyacin (mTOR), traditionally thought of as a gene prevalent in human cancers, has emerged in modern molecular medicine as an important contributing agent in angiogenesis and an important regulatory factor of angiogenic factors, such as NO. Acting mainly downstream of insulin signaling on the PI3 kinase, mTOR acts to further phosphorylate AKT and thus is a postitive effector of eNOS activation via phosphorylation [83]. Furthermore, inhibition of any kinase in this system, PI3K, mTOR, or AKT, has been shown to significantly reduce production of angiogenic actors, such as nitric oxide [83]. There is substantial evidence of impairment of this pathway in diabetes, either through disruption of Akt phosphorylation and expression [30] which then disrupts insulin secretion [31], and/or eNOS phosphorylation and activation [24]. Additionally, eNOS knockout mice reguarly show symptoms consistent with diabetes, including hyperlipidimia, hypertension and, perhaphs most importantly, insulin resistance [32]. This established association between type II diabetes and endothelial dysfunction through the PI3K/mTOR/AKT pathway provides insight into future supplementary treatments of diabetic patients that could be instrumental in lowering the CVD risk assocaited with the metabolic disorder.

AMP Kinase, nitric oxide and diabetes:  

AMP kinase, long standing as the energy gauge of the body (activated during times of high energy phosphate depletion), has emerged in recent years as a therapeutic agent for diabetic vascular disease. In fact, studies centered on AMPK activation have shown improved endothelial and smooth function [84].  The two leading diabetic drugs, namely metformin and rosiglitazone, show their metabolic effects partially through AMPK. These data, along with evidence from studies showing that chemical activation of AMPK in vivo with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) improves blood glucose concentrations and lipid profiles, make this enzyme an attractive pharmacological target for the treatment of Type II diabetes and other metabolic disorders. AMPK, addtionally, has been shown to increase eNOS phosphorylation, not unlike the PI3K/mTOR/AKT pathway [37,84]. eNOS activation conversly aides in AMPK modulation of glucose uptake via GLUT4 receptor translocation [38]. Restoring NO production through this pathway will likely provide benefit to diabetic patients and may even increase efficacy of existing drugs and prevent the unwanted cardiovascular side effects.

Nitrite restores nitric oxide homeostasis:  

Several studies now demonstrate that exogenous nitrite contributes to whole body NO production and homeostasis and is an alternate source of NO in vivo. Coadministration of nitrite with a NOS inhibitor for 3 weeks significantly attenuates hypertension [39]. Considerable published support for this theory derives from the following facts: NO produced from nitrite in the upper intestine is up to 10,000 times the concentrations that occur in tissues from enzymatic synthesis [40], nitrite can act as a circulating NO donor [41], can activate soluble guanylyl cyclase (sGC), change gene expression profiles [42], and nitrite can itself perform many actions previously attributable to NO [43] without the intermediacy of NO [42]. In fact, it has been shown that nitrite may be the endocrine mediator of NO based signaling [44]. Enhancing nitrite availability through therapeutic intervention by administering bolus nitrite prior to cardiovascular insult has shown remarkable effects in reducing the injury from myocardial infarction, ischemic liver and kidney injury, stroke and cerebral vasospasm [45-50] in animal models. It was recently revealed that nitrite, when given orally, is absorbed with greater than 95% bioavailability and is rapidly absorbed [51].

Nitrite seems to have residual effects long after it has been metabolized and cleared from the body by preconditioning the myocardium when given 24 hours prior to ischemic insult [52]. Nitrite has also been shown to augment ischemia induced angiogenesis and arteriogenesis [53]. Inhaled nebulized nitrite is a selective pulmonary vasodilator [54] and inhibits hypoxic and inflammatory pulmonary arterial hypertension [55]. Nitrite therapy has recently been shown to improve cardiac and neurological function and enhance survival after cardiac arrest [56]. Nitrite is also showing remarkable efficacy at promoting regional blood flow in sickle cell patients [57]. We now know that nitrite is just as efficacious when given orally at restoring NO biochemistry [58], reversing hypertension from NOS inhibition [39], protecting from myocardial ischemia-reperfusion injury [59] and even protecting from vascular inflammation, reversing endothelial dysfunction and reducing levels of C-reactive protein as a result of a high fat diet [60]. Most recently inorganic nitrate was shown to reverse features of metabolic syndrome in eNOS knockout mice through repletion of nitrite and NO homeostasis [3]. In humans, dietary nitrate and nitrite sources have been demonstrated to lower blood pressure [61-63] and decrease oxygen consumption during sub-maximal and maximal aerobic exercise [64, 65]. Collectively, these studies clearly reveal the benefits of nitrite as a means to restore or enhance NO bioavailability and/or homeostasis in an endothelium independent manner, offering a chance to correct NO homeostasis when endothelium derived NO is insufficient or dysfunctional as in diabetes.  Mice homozygous for the diabetes spontaneous mutation Leprdb become obese at 3-4 weeks of age. The mutant db/db mice are polyphagic, polydipsic, and polyuric. Thus, this mouse model recapitulates Type II diabetes well. Our preliminary data indicated that NO production was perturbed in db/db mice, and that provided justification for the use of nitrite as an alternate source of NO independent of endothelium derived NO. Providing exogenous nitrite likely improved insulin sensitivity and insulin signaling since less insulin was needed to maintain euglycemia and there was less weight gain in the diabetic mice.

Conclusion and future research considerations:  

This article has been dedicated to providing insight into the potential linking mechanisms between diabetes mellitus and cardiovascular disease. Endothelial dysfunction emerges as the major underlying link between the two, particularly through pathways affecting the activation of eNOS and further downstream, the bioavailabiity of NO.  

Therapautic efforts centered around restoring the eNOS system, either through the PI3K/AKT pathway, though regualtion of the mTOR gene, activation of the AMP kinase, or though dietary nitrite supplementation, provide a novel approach in reducing the burden of CVD in diabetic populations. Rescue of the system though L-Arginine supplementation, on the other hand, has not shown promising results, and thus negates a majority of the supplements avalabile through the retail channel today.


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