Angiotensin II (Ang II) is associated with hypertension, MS, IR, PD, and T2DM both systemically and at the local tissue level. Currently, there is evidence that a local tissue renin angiotensin aldosterone system (RAAS) is operative within the intima and islet as angiotensin type one (AT-1) receptors have been identified as being present on smooth muscle cells, endothelial cells, and the beta cells within the intima and islet 2021 Insulin is known to upregulate the AT-1 receptor 22 and there exists cross talk between the insulin and the Ang II signaling systems 23 In 1995, Copper et al. were able to demonstrate that amylin activates the RAAS with elevations in renin and aldosterone in humans 24 and, in 2001, Ikeda et al. were able to demonstrate that insulin, proinsulin and amylin infusions resulted in significant increases in renin release and that proinsulin and amylin enhanced this insulin-stimulated renin release in the perfused rat kidney 25.

Taken together, these data support the strong influence of a local RAAS mechanism operating within the intima and islet for the local production of excess Ang II. The islet is quite vascular with an abundant supply of intra islet capillaries and endothelial cells and the intima is lined by a continuous monocellular layer of endothelial cells (additionally, the arterial vessel wall becomes highly vascular through the process of plaque angiogenesis as the vulnerable plaque evolves during the process of atheroscleropathy).

This allows the vascular NAD(P)H oxidase enzyme to come into play. Ang II is one of the most potent endogenous stimuli for the generation of superoxide O₂^- via the activation of vascular NAD(P)H oxidase. 2627

The interruption of this mechanism by the angiotensin converting enzyme inhibitor (ACEi) ramipril in the Heart Outcomes Prevention Evaluation (HOPE) study may help to explain the 32% risk reduction for developing T2DM as well as the dramatic reduction in cardiovascular events. 28

A special reference to Griendling and Harrison seems appropriate: "Out, damned DOT! Out I say" (where the damned DOT represents the unpaired dots on Lewis diagrams). 29 One of the best ways to prevent these dots from forming is to prevent excess substrates (table 3) which cause the multiple toxicities and the multiplicative effect of the A-FLIGHT toxicities associated with MS, IR, PD, and T2DM.

In MS, IR, PD, and T2DM the intima and islet milieu will be laden with the necessary substrates (hyperinsulinemia, hyperproinsulinemia and hyperamylinemia) to activate the damaging cascading mechanism of Ang II, NAD(P)H oxidase, superoxide (O₂^-) and peroxynitrite (ONOO^-) production while consuming the natural endogenously produced antioxidant nitric oxide (NO) within the vulnerable intima and islet.

Advanced glycosylation endproducts (AGEs) are formed as a result of the non-enzymatic damaging protein glycation due to an excess of glucose (hyperglycemia) present in both T1DM, PD, and T2DM. AGEs are initially formed through the process of a glucose nucleophilic addition reaction with proteins forming a Schiff base followed by the formation of an Amadori compound which undergoes further reactions, rearrangements, dehydrations and cleavage resulting in brown insoluble, cross linked complexes called AGEs. This process is thought to liberate H₂O₂ through two pathways: the first is the 1,2-enolization pathway which leads to 3-deoxyglucosone forming H₂O₂ and glucosone; the second pathway is the 2,3-enolization pathway leading to 1-deoxyglucosone and putative 1,4-deoxyglucosone. Under oxidative conditions, the 2,3-enediol is thought to generate H₂O₂ and carboxymethyllysine. 3-deoxyglucosones are known to be both highly reactive intermediates in non-enzymatic glycosylation and also potent cross-linkers which are responsible for the polymerization of proteins to AGEs. These highly cross-linked proteins, especially collagen, cause a stiffening within the vessel which results in decreased compliance of the arterial vessel wall and may well play an important role in the development of diabetic diastolic dysfunction, diabetic cardiomyopathy, and the diastolic dysfunction of the arterial vessel wall. Furthermore, there are advanced fructosylation endproducts (AFEs), which actually have a greater affinity binding to proteins than glucose and follow a similar pattern in the production of the ROS. 30313233 An excellent in depth review of AGE can be found in an article by Aronson and Rayfield where they discuss how hyperglycemia promotes atherosclerosis 34.

The multiligand immunoglobulin superfamily cell surface receptor: the receptor for advanced glycation endproducts (RAGE) is up-regulated by the presence of AGE and results in the signal transduction of nuclear factor kappa B (NFkappa B) which then results in a chronically active inflammatory state and links this section to section (I). Inflammation Toxicity and atheroscleropathy. 3536

Ageing has been shown to be associated with an increased risk of developing T2DM and atheroscleropathy. Ageing allows the multiplicative effects of the A-FLIGHT toxicities to become manifest. Advanced ageing leads to impaired endothelial nitric oxide synthesis and also enhanced endothelial apoptosis.

In addition, aged cells have a significantly enhanced concentration (more than 3 fold) of oxidized low density lipoprotein, TNFalpha and caspase-3 activity as compared to young cells. The decrease in eNO associated with aged cells creates a deficiency of the naturally occurring antioxidant eNO. 54

Similarly, excess redox stress is felt to contribute to ageing. Information on the relationship of redox stress and ageing and inflammation (see section "(I). Inflammation Toxicity") is rapidly increasing and gaining wider recognition. 5556

Amylin, also termed islet amyloid polypeptide (IAPP) is a 37 amino acid polypeptide co-synthesized, co-packaged, and co-secreted by the islet beta cell with insulin. It may be considered insulin's a fraternal twin.

Amylin parallels insulin synthesis, secretion, and excretion so that whenever you have hyperinsulinemia you have hyperamylinemia and, in the same way, when insulin levels decline amylin levels decline.

Amylin stimulates lipolysis in vivo and may be a possible mediator of induced insulin resistance. Ye et al., were able to demonstrate that amylin infusion (5 nmol/h for 4 h) conscious rats that fasted for 5–7 hours resulted in an elevation of insulin, lactate and glucose (P < 0.05 vs. control).

Despite the rise in insulin, plasma non-esterified fatty acid and glycerol were also elevated (P < 0.001). Although the plasma triglyceride content was unaltered, the triglyceride content in the liver was increased by 28% (P < 0.001) with a similar tendency in muscle (18%, P = 0.1). These effects were blocked by the rat amylin antagonist amylin-(8–37) and also by the anti-lipolytic agent acipimox. The authors concluded that amylin could exert a lipolytic-like action in vivo. 57

This elevation in amylin would correspond to the insulin resistant state with associated elevation in amylin in humans. These data indicate that amylin may play a role by elevating free fatty acids which would aggravate or induce the underlying insulin resistance and provide a mechanism for increasing the free fatty acid substrate for increased redox stress, cytotoxicity and intimal remodeling associated with atheroscleropathy.

There are amylin binding sites within the renal cortex and amylin activates the RAAS with elevations in renin and aldosterone 24

These findings suggest that glucotoxicity resulting in AGE formation both promotes and accelerates redox stress. 58

Janson et al. have found that intermediate sized toxic amyloid particles (ISTAPs) have been found to be cytotoxic to beta cells inducing apoptosis by membrane disruption. 59

Once atherosclerosis and atheroscleropathy has been initiated and sustained, this process is self – perpetuating (self sustaining) and the vicious cycle of ROS begetting ROS comes into play with all of its inherent complications. It's as if the multiple A-FLIGHT toxicities polymerize with redox stress and its associated ROS acting as an accelerant. The associated ischemic cardiomyopathy and the distinct diabetic cardiomyopathy are not within the scope of this review.

Free fatty acid (FFA) elevation is known to be associated with IR, MS, PD, and T2DM. The metabolically active form of FFAs are cytosolic long-chain acyl-CoA esters (LCACoA) and are responsible for cytosolic neutral triglyceride deposition in adipose and non-adipose tissues.

In 2001, McGarry gave an excellent presentation at the American Diabetes Association meeting (ADA 2001 Banting Lecture), discussing in detail how toxic FFA and LCACoA may be important in the development of insulin resistance, progressive beta cell dysfunction and death associated with T2DM. 64

Central obesity is associated with increased cytosolic neutral fat triglyceride stores in adipose and non-adipose tissues such as muscle (skeletal and cardiac), the liver, pancreatic beta cells and, possibly, endothelial cells. 6465

Intra-myocellular lipid was found to be more highly correlated with insulin resistance than any other commonly measured indices such as body mass index, waist-to-hip ratio or total body fat. Low insulin sensitivity was accompanied by a marked increase in intra-myocellular lipid. Bakker et al.65 proposed that the chronic low-grade production of ROS produced by respiring mitochondria is enhanced by excessive cytosolic triglyceride stores and LCACoA esters in non-adipose tissue.

They proposed that LCACoA esters exert an inhibitory effect on the adenosine nucleotide translocator with a resultant decrease in the ADP available. This decrease in ADP slows the flow of electrons along the electron transfer chain and increases the possibility of having single unpaired electrons to create the superoxide anion (O₂^-) increasing oxidative mitochondrial stress, thus resulting in a dysfunctional cell. Moreover, they suggest that these phenomena not only accelerate the atherosclerotic process but also induce endothelial dysfunction and microalbuminuria prior to the development of T2DM and possibly beta cell dysfunction and failure. 65

It is difficult to completely separate FFA toxicity from the sections which follow on lipoprotein toxicity and triglyceride toxicity as there is a dynamic relationship between these three in the A-FLIGHT toxicities. In fact, FFAs are transported by the protein fraction, albumin, and lipases are constantly removing the long chain fatty acids from the glycerol backbone of triglycerides at the interface of the capillary endothelial cells creating free fatty acids which can freely move into cells throughout the body. Intracellularly, the FFAs are then added to the glycerol backbone in order to form cytosolic triglycerides stored as neutral fat, or are oxidized for fuel and energy generating ATP. If mitochondrial beta oxidation is over utilized or dysfunctional, the excess may then undergo the toxic non-beta non-mitochondrial pathway generating toxic FFAs or ceramide (see section "(L). Lipotoxicity – Specific").

Lipotoxicity promotes oxidative stress and is associated with MS, IR, PD, and T2DM. There is an associated defect of lipoprotein metabolism frequently referred to as the "lipid triad". Elevated VLDL or triglycerides, atherogenic small dense LDL, and decreased HDL comprise this triad which is associated with atheroscleropathy and coronary heart disease as well as increased redox stress. 666768

The increased VLDL, triglycerides, atherogenic small dense LDL cholesterol and the diminished amount of the anti-atherogenic, antioxidant anti-inflammatory high density lipoprotein cholesterol would reduce the natural antioxidant reserve. This combination supports an increase in redox stress in addition to the previously discussed FFA toxicity. This also tends to support the oxidation, glycation and glycoxidation of existing lipoproteins (modification) which results in increased ROS and redox stress.

Lipoproteins have the function of transporting lipids throughout the body. Low density lipoproteins are responsible primarily for the transport of cholesterol with the protein moiety involved: apolipoprotein (Apo) B 100. Very low density lipoproteins are responsible for the transport of triglycerides with the protein moiety involved: Apo E. High density lipoproteins are responsible for reverse cholesterol transport and play an important role in being a naturally occurring potent anti-inflammatory and antioxidant agent with the protein moiety involved: Apo A. It is the protein moiety of the lipoproteins that is modified by the processes of oxidation, glycation, and glycoxidation with a resultant increase in redox stress and the production of ROS. Furthermore, the modification of the protein moiety is responsible for their retention within the intima, inducing atherogenesis and thus atheroscleropathy. 6970

Lipotoxicity is also associated with MS, IR, PD, and T2DM. Unger et al.717273747576 feel this specific lipotoxicity is attributed to products of the excessive non-beta- (non-mitochondrial) oxidative metabolism of FFA in the skeletal and the myocardial muscle, the liver and the pancreatic islets.

In addition, these toxic metabolic products are thought to cause the complications of MS, IR, PD, and T2DM by creating cellular dysfunction and, in time, promoting programmed cellular death (lipoapoptosis). 7475 In the normal state, FFA delivery to non-adipose tissue is closely regulated to its need for fuel. FFAs normally rise during exercise and fasting in order to meet metabolic requirements and thus, homeostasis is maintained. However, as a result of over-nutrition (western diet), the FFA influx may exceed FFA usage and FFA non-beta oxidation ensues.

These non-mitochondrial FFA metabolites, which are responsible for injuring cells, result in lipoapoptosis, include triglycerides, ceramide, and products of lipid peroxidation. Ceramide (an amino alcohol with a LCACoA attached to the amino group) has been implicated for some time in the apoptotic pathway of the T1DM autoimmune destruction of beta cells by sphingomyelin degradation. 77

Ceramide can be formed in these cells by direct de novo synthesis from FFAs. 72 Ceramide is responsible for the induction and activation of NFkappa B. 78

In the process of developing T2DM, only those beta cells with the highest fat content give way to the ceramide cascade thus leaving enough functioning beta cells to maintain insulin independence but not enough to compensate for the co-existing insulin resistance with the subsequent development of impaired glucose tolerance, impaired fasting glucose and the development of overt T2DM. This entire process is magnified and progresses due to an intense redox (oxidative stress within the islet and intima which incorporates and implicates the multiplicative manifold A-FLIGHT toxicities).

Insulin toxicity (hyperinsulinemia, hyperproinsulinemia, and hyperamylinemia) is associated with MS, IR, PD, and early T2DM. In late T2DM as beta cell failure develops there is no longer insulin toxicity. Insulin is known to up-regulate the number of AT-1 receptors, activate the RAAS, and be capable of cross talking with the AT-1 receptor. Recently, AT-1 receptors have been identified on the islet beta cell and the islet endothelial cell.

Thus, hyperinsulinemia can be linked back to the section "(A). Angiotensin II" with resultant increased redox stress systemically as well as within the intima and islet as insulin, proinsulin and amylin are all three elevated within the intima and islet milieu. 202122232425

Endogenous Hyperinsulinemia (eHI) is associated with MR, IR, PD, and early T2DM. Additionally, eHI is associated with hypertension and atheroscleropathy (coronary artery disease). eHI is also associated with elevated FFA, plasminogen activator inhibitor-1 (PAI-1), elevated sympathetic tone and activity, increased sodium and water reabsorption leading to volume expansion which leads to and supports hypertension in the clustering phenomenon of MS relating to section (H). Hypertension toxicity. Previously discussed, insulin, proinsulin, and amylin have been noted to contribute to the elevation of Ang II with increases in renin and aldosterone.

Amylin the fraternal twin of insulin has been shown to induce lipolysis which elevates FFA and links to the sections on (F), (L), and (T). The reader will note that the various sections within the A-FLIGHT toxicities interact and play off one another creating a vicious cycle of promoting redox stress within the intima and islet.

Additionally, it is important to note that increased proinsulin concentrations predict death and morbidity caused by coronary heart disease independent of other major cardiovascular risk factors. 7980

A new insight into the study of atherosclerotic plaques has evolved over the past decade and now the accepted role of inflammation in vulnerable plaque pathology has been widely accepted in the field of atherosclerology.

Currently, chronic inflammation is gaining momentum as a prelude to MS, IR, PD, and T2DM.

Increasingly, this quartet in the continuum of the natural history of diabetes is being accepted by diabetologists and researchers as a chronic inflammatory disease. 9818283848586878889909192

The four "cardinal signs" of inflammation described by Aulus C. Celsus in De re medicina in 30 A.D. are: rubor, calor, dolor, and tumor and currently there is a large body of information that atherosclerotic vulnerable plaques (VP) fit the above criteria.

Glucotoxicity is associated with both type 1 and type 2 diabetes mellitus and, thus, the similarly shared multiple opathies associated strongly with redox stress (figure 7). There is a major difference between T1DM and T2DM in regards to atheroscleropathy. In T1DM the atheroscleropathy does not start until there is a diagnosis and glucotoxicity develops acutely. In contrast T2DM is preceded by 5–10 years of a MS, IR, and PD state consisting initially of postprandial glucose elevations (IGT) then fasting glucose elevation.(IFG). Additionally, MS and IR comes with the attendant A-FLIGHT toxicities. (table 3)

Four subsections are important in this discussion.

Hypertension is associated with increased redox stress and ROS activity. Furthermore, hypertension is associated with ROS mediated vascular damage and is closely associated with the activation of Ang II (see section (A). Angiotensin II) and its effect on the vascular NAD(P)H oxidase superoxide (O₂^-) generating enzyme. 26

Cellular sources of vascular superoxide production are the endothelial cell, vascular smooth muscle cell and adventitial fibroblasts. The major enzymatic sources are NAD(P)H oxidase, xanthine oxidase and, paradoxically, the eNOS enzyme (in the presence of oxidative stress or deficiency of L-arginine or tetrahydrobiopterin).114

It is important to note that glucotoxicity is closely associated with activation of the RAAS at the local, interstitial and tissue levels.

Recently, amylin has been implicated as being elevated in patients who have a positive family history associated with hypertension and is elevated prior to the onset of hypertens