These pages are best viewed with Netscape version 3.0 or higher or Internet Explorer version 3.0 or higher. When viewed with other browsers, some characters or attributes may not be rendered correctly.

ORIGINAL ARTICLE

Theoretical Mechanisms by Which Hyperglycemia and Insulin Resistance Could Cause Cardiovascular Diseases in Diabetes

George L. King, MD
Hisao Wakasaki, MD, PHD

Multiple metabolic factors have been shown to increase the risks of atherosclerosis in diabetic patients. Among these factors, hyperglycemia and insulin resistance are two metabolic abnormalities that are specific in their association to the diabetic state. A large number of studies have been done to determine the possible mechanisms by which these two metabolic changes can cause an enhancement of the atherosclerotic process. In this review, we address the possible mechanisms at the biochemical level by which hyperglycemia could be causing its adverse effects in the vascular cells. In addition, we also describe a relatively new concept suggesting that insulin at the physiological level has important anti-atherogenic actions. The loss of insulin action in insulin-resistant or insulin-deficient states predisposes to the vasculature of atherosclerosis.

The results of the Diabetes Control and Complications Trial clearly established hyperglycemia as the major causal factor for the development of diabetic microvascular complications (1). A role for hyperglycemia as an independent risk factor for the development of cardiovascular disease is supported by the United Kingdom Prospective Diabetes Study (2). Nevertheless, it is likely that hyperglycemia is contributing directly or indirectly to the atherosclerotic process, because glycemic control can delay the onset or progression of nephropathy, which is an independent risk factor of atherosclerosis (1).

Figure 1—Summary of changes in the types of vascular cells and extracellular matrix in the retina, renal glomeruli, and arteries of diabetic patients as compared with nondiabetic patients.

Although systemic factors such as hyperglycemia clearly play an important role in the development of diabetic complications, it should be noted that specific tissue responses or local factors might be just as important as systemic factors. The importance of tissue-specific responses or factors are clearly demonstrated by the differences in the changes of vascular cells in the retina, renal glomeruli, and arteries, as shown in Fig. 1. For example, in diabetic retinopathy, the retinal capillary endothelial cells have the propensity to proliferate, as shown by the formation of microaneurysms and neovascularization, whereas in diabetic nephropathy and cardiovascular disease, endothelial cells are either damaged or lost excessively. For the vascular contractile cells, retinal capillary pericytes—the counterpart of arterial smooth muscle cells—are lost, whereas in diabetic macrovascular diseases, arterial smooth muscle cells proliferate excessively as in atherosclerosis. These pathological changes clearly suggest that localized tissue responses to hyperglycemia are just as critical as the systemic factors. In addition, the pathological findings in the microvasculature have clearly established that diabetic vascular lesions in retinopathy and nephropathy are not due to acceleration of aging, because the classic lesions of renal mesangium expansion and retinal pericyte loss are not typically observed in these organs in the aging process. In the cardiovascular tissues, however, many of the atherosclerotic lesions in diabetic patients are qualitatively comparable to those observed in nondiabetic patients, except that the severity of lesions appears to be accelerated (3). Therefore, for the molecular and biochemical mechanisms describing the adverse effects of hyperglycemia to be valid, they have to explain how hyperglycemia can cause different vascular pathologies in the various tissues.

Several biochemical mechanisms appear to explain the adverse effects of hyperglycemia on vascular cells, which may accommodate most of the data produced in this area (Table 1). However, a single theory that can explain all the vascular changes has not been established, probably because the metabolites of glucose can affect numerous cellular pathways, both extra- and intracellularly.

Glucose can react extracellularly in nonenzymatic reactions, in which primary amines of amino acids in proteins form glycated compounds or oxidants. These can secondarily act on inflammatory cells to release cytokines or directly act on vascular cells to cause vascular dysfunctions (4).

Increased amounts of glucose can also be transported intracellularly—mostly by glucose transporters, GLUT-1 and possibly GLUT-4—and metabolized to increase flux through the sorbitol pathway, change redox potential, or alter signal transduction pathways, such as the activation of diacylglycerol (DAG) and protein kinase C levels (PKC) (4–7). It is possible that the common pathway by which all the intra- and extracellular changes induced by hyperglycemia are mediating their adverse effects is the alteration of various signal transduction pathways, such as the activation of DAG-PKC. The activation of the PKC pathway can, in vascular cells, regulate permeability, contractility, extracellular matrix, cell growth, angiogenesis, cytokine actions, and leukocyte adhesions, all of which are abnormal in diabetes. In the first part of this article, the available data supporting a role for the activation of DAG-PKC in the development of diabetic vascular complications are reviewed.

MECHANISMS OF HYPERGLYCEMIA-INDUCED PKC ACTIVATION— The PKC family includes at least 11 isoforms (, 1, 2, , , , , , , , µ), representing the major targets for lipid second messengers (8). Cellular DAG, which is the physiological activator of PKC, can be derived from multiple sources, including the hydrolysis of phosphatidylinositides (PIs), the metabolism of phosphatidylcholine by phospholipase C or D, or de novo synthesis.

Increases in total DAG contents have been demonstrated in a variety of tissues associated with diabetic vascular complications, including the retina (15), aorta, heart (16), and renal glomeruli (17,18) of diabetic animals and patients (9,10). In addition, DAG levels have also been reported to be increased in classic "insulin-sensitive" tissues, such as the liver and skeletal muscle, in diabetic animals. Increasing glucose levels from 5 to 22 mmol/l in the media elevated the cellular DAG contents in all cultured vascular cells studied. The increases in DAG-PKC may not occur immediately but reach maximum in 3–5 days after elevating glucose levels and chronically persisting in the elevated levels. In fact, Inoguchi et al. (11) reported that euglycemic control by islet cell transplant after 3 weeks was not able to reverse the increases in DAG or PKC levels in the aorta of diabetic rats. These results clearly suggested that the activation of DAG-PKC could be sustained chronically.

Although cellular DAG contents can be increased by agonist-stimulated hydrolysis of PI, this mechanism is most likely not involved in diabetes, as inositol phosphate products were not found to be increased by hyperglycemia in aortic cells and glomerular mesangial cells (9,10).

The activation of PKC by hyperglycemia may be tissue specific, because it has been noted in the retina, aorta, heart, and glomeruli but may not occur in the brain and peripheral nerves (9,10). Similar increases in DAG and PKC levels have also been shown in multiple types of cultured vascular cells when glucose levels were increased (9,10). Of the various PKC isoforms in vascular cells, PKC- and - isoforms appear to be preferentially activated by immunoblotting studies in the aorta and heart of diabetic rats (11) and in cultured aortic smooth muscle cells (28) exposed to high levels of glucose. However, increases in multiple PKC isoforms were noted in some vascular tissues, such as PKC-, -2, and - isoforms in the retina. PKC-, -1, and - isoforms in the glomeruli of diabetic rats have also been reported (9–12).

As stated earlier, it is not surprising that glucose and its metabolites can cause many cellular parameters to change, as it is the main source of fuel in many cells. However, for a hyperglycemia-induced change to be credible as a causal factor of diabetic complications, it has to be chronically altered, be difficult to reverse, cause similar vascular changes when activated without diabetes, and be able to prevent complications when it is inhibited or removed. Evidence concerning the DAG-PKC activation to fulfill the first two criteria has been presented. In the following, supporting data are discussed that suggest that the last two criteria are beginning to be accumulated.

CELLULAR AND FUNCTIONAL ALTERATIONS IN VASCULAR CELLS INDUCED BY DAG-PKC ACTIVATION— Multiple cellular and functional abnormalities in the diabetic vascular tissues have been attributed to the activation of DAG-PKC pathways. Some of these adverse changes in the vascular cells or tissue are schematically described in Fig. 2.

Figure 2—Schematic diagram of the adverse effects of hyperglycemia in vascular cells. This is a potential mechanism by which DAG-PKC activation can cause vascular cellular dysfunctions and pathologies induced by advanced glycation products (AGEs), oxidants, or directly by hyperglycemia. ANP, atrial natriuretic peptide; ET-1, endothelin-1; ICAMS, intercellular adhesion molecules; PAI-1, plasminogen activator inhibitor-1; Vit. E, vitamin E.

N⁺, K⁺ ATPase
Na⁺, K⁺ ATPase, an integral component of the sodium pump, is involved in the maintenance of cellular integrity and functions such as contractility, growth, and differentiation (6). It is well established that Na⁺, K⁺ ATPase activity is generally decreased in the vascular and neuronal tissues of diabetic patients and experimental animals (6). However, the mechanisms by which hyperglycemia inhibits Na⁺, K⁺ ATPase activity have provided some conflicting results regarding the role of PKC.

Phorbol esters, activators of PKC, have been shown to prevent the inhibitory effect of hyperglycemia on Na⁺, K⁺ ATPase (6), which suggested that PKC activity might be decreased in the diabetic milieu. Yet, we recently reported that an elevated glucose level (~20 mmol/l) increased PKC and cytosolic phospholipase A₂ (cPLA₂) activities, resulting in increases of arachidonic acid release and prostaglandin E₂ (PGE₂) production and decreases in Na⁺, K⁺ ATPase activity. Inhibitors of PKC or PLA₂ prevented hyperglycemia-induced reduction in Na⁺, K⁺ ATPase activities in aortic smooth muscle cells and mesangial cells (13). The apparent paradoxical effects of phorbol ester and hyperglycemia in the enzymes of this cascade are probably due to the quantitative and qualitative differences of PKC stimulation induced by these stimuli. Phorbol ester, which is not a physiological activator, probably activated many PKC isoforms and increased PKC activity by 5–10 times, whereas hyperglycemia can only increase PKC activities by twofold, a physiologically relevant change (13) that affected selective PKC isoforms. Thus, the results derived from the studies using phorbol esters are difficult to interpret with respect to their physiological significance.

Extracellular matrix components
Thickening of capillary basement membrane and increases in extracellular matrix are two of the early structural abnormalities observed in almost all tissues, including the vascular system, in diabetes. Because basement membrane can affect numerous functions—such as structure support, vascular permeability, cell adhesion, proliferation, differentiation, and gene expressions—alterations in its components may cause vascular dysfunctions.

Histologically, increases in type IV and VI collagen and fibronectin and decreases in proteoglycans are observed in the mesangium of diabetic patients. Increases in these proteins can be replicated in mesangial cells incubated in increasing glucose levels (5–20 mmol/l), which were prevented by general PKC inhibitors (14–16). As described above, the increased expressions of transforming growth factor- (TGF-) have been implicated in the development of mesangial expansion and basement membrane thickening in diabetes. Ziyadeh and colleagues (15) reported that neutralizing TGF- antibodies significantly reduced collagen synthesis and gene expression of Type 1 (IV) collagen and fibronectin in the renal cortex of diabetic rats and cultured mesangial cells exposed to high glucose levels. Considering that PKC activation can increase TGF- expression and the production of extracellular matrix (ECM), it is not surprising that several reports have shown that PKC inhibitors can also prevent hyperglycemia- or diabetes-induced increases in ECM and TGF- in mesangial cells or renal glomeruli (12).

Vascular blood flow
Abnormalities in vascular blood flow and contractility have been reported in many organs of diabetic animals and patients, including the kidney, retina, peripheral arteries, skin, and microvessels of peripheral nerves. In the retina of diabetic patients and animals with a short disease duration and without clinical retinopathy, retinal blood flow has been shown to be decreased (5). However, retinal blood flow may be normal or increased with longer duration of retinopathy (5). Multiple lines of evidence support that the decreases in retinal blood flow are due to PKC activation. For example, introduction of a PKC agonist, such as phorbol ester, into the retina decreases retinal blood flow (5). Decreases in retinal blood flow in diabetic rats have been reported to be normalized by PKC inhibitors (5). In addition to what has been reported in the retina, decreases in blood flow have also been reported in the peripheral nerves of diabetic animals, which can be normalized by PKC inhibition (5).

Abnormalities in hemodynamics also have been clearly documented to precede diabetic nephropathy (17). Elevated renal glomerular filtration rate and modest increases in renal blood flow are characteristic findings in type 1 diabetic patients and experimental diabetic animals. Diabetic glomerular hyperfiltration is likely to be the result of hyperglycemia-induced decreases in arteriolar resistance, especially at the level of the afferent arteriole, resulting in an elevation of glomerular filtration pressure. Multiple mechanisms have been proposed to explain the increases in glomerular filtration rate and filtration pressure, including an enhanced activity of angiotensin (18) and prostinoid production. It is possible that the activation of DAG-PKC may also play a role in the enhancement of angiotensin actions, considering that angiotensin mediates some of its activity by the activation of the DAG-PKC pathway. In addition, increases in vasodilatory prostanoids, such as PGE₂ and PGI₂, could also be involved in causing glomerular hyperfiltration in diabetes. The enhanced production of PGE₂ induced by diabetes and hyperglycemia could be the result of sequential activation of PKC and cPLA₂, a key regulator of arachidonic acid synthesis (13).

In the microvessels, increases in the activities of nitric oxide (NO), a potent vasodilator, may also enhance glomerular filtration. Urinary excretions of NO₂ /NO₃, stable metabolites of NO, have been reported to be increased in diabetes of short duration (19), possibly due to enhanced expression of inducible NO synthase (iNOS) gene and increased production of NO in mesangial cells. In addition, increases in both iNOS gene expression and NO production can be mimicked by PKC agonist and inhibited by PKC inhibitors when induced by hyperglycemia (20), suggesting that NO production might be increased in diabetes through PKC-induced iNOS overexpression. Thus, PKC can regulate renal hemodynamics by increasing or decreasing NO production depending on the cell type and tissue location.

In the macrovessels, increases in contractility observed in diabetes are due to a resistance in the relaxation responses after contraction induced by cholinergic agents (19). These abnormal responses can also be prevented by PKC inhibitor (20), suggesting that PKC activation may play a general role in causing abnormal peripheral hemodynamics in diabetes.

Vascular permeability and neovascularization
Increased vascular permeability is a characteristic systemic vascular abnormality in diabetic animals, where increased permeability can occur at as early as 4–6 weeks' duration of diabetes, suggesting endothelial cell dysfunctions. PKC activation can directly increase the permeability of albumin and other macromolecules through barriers formed by endothelial cells (22), probably by phosphorylating the cytoskeletal proteins forming the intracellular junctions. Interestingly, phorbol ester–induced increases in endothelial permeability may also be regulated by PKC-1 activation (23), which is consistent with the preferential activation of PKC- isoforms in diabetes.

PKC activation can also regulate vascular permeability and neovascularization via the expression of growth factors, such as the vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), which is increased in ocular fluids from diabetic patients and has been implicated in the neovascularization process of proliferative retinopathy (24). We reported that both the mitogenic and permeability-inducing actions of VEGF/VPF are partly due to the activation of PKC- via the tyrosine phosphorylation of phospholipase C-. Inhibition by the PKC- selective inhibitor LY333531 can decrease endothelial cell proliferation, angiogenesis, and permeability induced by VEGF (25). In addition, Williams et al. (26) showed that the expression of VEGF was increased in aortic smooth muscle cells by elevating the glucose concentration and was inhibited by PKC inhibitors.

USE OF PKC-ISOFORM SELECTIVE INHIBITOR — Chronic studies involving PKC inhibitors such as staurosporine, H-7, and GF109203X have not been possible because of their toxicity, which is the result of their nonspecific effects on other kinases (21) and general inhibition of all PKC isoforms. Because analysis of retina, kidney, and cardiovascular tissues of diabetic rats showed that PKC- isoforms were preferentially activated (9,11,12), a specific inhibitor for PKC- isoforms should be more effective and less toxic than a nonisoform-specific PKC inhibitor.

Recently, we reported that abnormal retinal and renal hemodynamics and the increases in albuminuria in diabetic rats can be ameliorated by an orally available PKC- isoform selective inhibitor LY333531 in parallel with the inhibition of diabetes-induced PKC activation in retina and renal glomeruli (21). LY333531 prevented the overexpression of TGF-, 1(IV) collagen, and fibronectin in the renal glomeruli of diabetic rats (12). In addition, treatment with LY333531 was able to normalize neuronal blood flow and nerve conduction velocity in diabetic rats (N. Hotta, personal communication). These results again suggested that activation of PKC- isoforms is involved in the development of some early abnormalities of diabetic vascular complications. PKC inhibitors could also mediate their effects by the inhibition of angiotensin actions, which may be increased, as ACE inhibitors have been proven to delay the progression of nephropathy (27). However, long-term studies are still needed to clarify the usefulness of LY333531 to prevent the chronic pathological changes of diabetic vascular complications.

VITAMIN E— Recent studies using antioxidants have provided some support that oxidative stress is increased in the diabetic state (7). Vitamin E, a well-studied antioxidant, has the additional interesting property of inhibiting the activation of DAG-PKC in vascular tissues and cultured vascular cells exposed to high glucose levels (28–30). Vitamin E can inhibit PKC activation, probably by decreasing DAG levels (28–30), as the direct addition of vitamin E to purified PKC- or - isoforms in vitro did not have any inhibitory effect. Recently, the activation of DAG kinase has been suggested to be one potential site of action for vitamin E to inhibit PKC, because DAG kinase metabolizes DAG to phosphatidic acid, thus attenuating PKC activity (31).

Biochemically, intraperitoneal injections of vitamin E prevented the increases in both DAG levels and PKC activities in the retina, aorta, heart, and renal glomeruli of diabetic rats (28–31). Functionally, vitamin E treatment prevented the abnormal hemodynamic properties in the retina and kidney of diabetic rats in parallel with the inhibition of DAG-PKC activation. In addition, increased albuminuria was prevented by vitamin E treatment in diabetic rats. Thus, it is possible that some of the PKC activation induced by diabetes could also be the result of excessive oxidants, which are known to activate PKC and can be produced by hyperglycemia, leading to the development of the vascular dysfunctions in early stages of diabetes (28–31).

CARDIOVASCULAR COMPLICATIONS— Multiple studies have reported that a host of cardiac dysfunctions can occur in diabetic rats and in diabetic patients, even those with only 2–3 years of disease, suggesting that hyperglycemia can cause myocardium dysfunctions directly (32). We also reported that the diabetic state induces the activation of PKC- isoform in the heart of rats (11). To determine whether activation of PKC- isoform can cause cardiac abnormalities, we made transgenic mice overexpress PKC-2 isoform specifically in the myocardium by the use of tissue-specific promoter myosin heavy chain-. These mice developed cardiac hypertrophy, cardiomyocytes injuries, and fibrosis at 8–12 weeks of life. At the 20th week, cardiac atrophy and severe fibrosis were observed (33). Treatment with a PKC- isoform inhibitor (LY333531) prevented most of the functional and pathological changes in the hearts of the transgenic mice, clearly demonstrating that excessive PKC- activation can cause cardiomyopathy.

INSULIN RESISTANCE— The second major risk factor, which is specific for patients with diabetes or glucose intolerance, is abnormalities of insulin actions in the vascular tissues. Both insulin resistance and, possibly, hyperinsulinemia have been suggested as risk factors for the development of cardiovascular complications in diabetes. Because the epidemiological studies have been reviewed elsewhere in this supplement, we will not discuss them here (34). In this article, we provide a brief review of the recent hypothesis that insulin at the physiological level has anti-atherogenic actions, whereas in insulin-resistant or hyperinsulinemic conditions, a state of enhanced atherogenesis could occur (Fig. 3).

Figure 3—Schematic drawing of insulin's signaling pathways in vascular smooth muscle cells. The drawing suggests that the activation of either PI3 kinase or MAP kinase (MAPK) can mediate insulin's actions, with the former stimulating mainly anti-atherogenic effects and the latter stimulating atherogenic actions. The diminution of insulin's effect of P13 kinase in the vascular cells can lead to an enhancement of atherosclerosis. Def, deficient; INS, insulin; IR, insulin receptor; Resist, resistant; SMC, smooth muscle cells.

Recent reports have clearly established that vascular cells are capable of responding to insulin with a whole range of action (35). In the past, the focus of insulin's actions on the vasculature has been on its mitogenic effects, mainly on the vascular smooth muscle cells (36). It is believed that hyperinsulinemia can contribute to the acceleration of atherosclerosis by enhancing the proliferation of aortic smooth muscle cells and increasing the synthesis of the extracellular matrix proteins in the arterial wall. Evidence in support of this comes from studies using culture aortic or arterial smooth muscle cells, which can be stimulated to proliferate by insulin. However, the mitogenic actions of insulin on smooth muscle cells are quite weak and may not be significant in physiological conditions (37). Most of the studies have shown that insulin can only stimulate the growth of arterial smooth muscle cells at concentrations >10 nmol/l. In the insulin-resistant or hyperinsulinemic state, the plasma level of insulin rarely reached and sustained these levels for a significant period of time. However, we and others have proposed that insulin may exert its atherogenic actions on the smooth muscle cells by enhancing the mitogenic actions of more potent growth factors, such as platelet-derived growth factor or insulin-like growth factors (37). Even if insulin can enhance the atherogenic actions of other growth factors, that still does not explain the apparent contradictions to the hypothesis: vascular cells are not resistant to insulin, whereas adipose tissues and skeletal muscle are. Therefore, inherent in the hypothesis stated above is that there is tissue specificity with respect to insulin-resistant states. However, there is very little evidence to support the notion that vascular tissues are protected against insulin resistance. In fact, recent studies on insulin's vasodilatory effects would support that insulin resistance is present at the arterial levels. Specifically, insulin has been postulated to enhance NO production, either through acute activation of NOS or through enhanced expression of endothelial cell nitric oxide synthase (eNOS) (38). Several laboratories have suggested that this action of insulin is blunted in the insulin-resistant state. Furthermore, the necessity of hyperinsulinemia being present for the development of atherosclerosis is also questioned by the epidemiological evidence that type 1 diabetic patients also suffer from an accelerated rate of cardiovascular disease even though they do not have sustained levels of hyperinsulinemia. In addition, correlation of hyperinsulinemia to atherosclerosis is not always found in non-Caucasian populations (39).

Because of these conflicting results, we propose the following theory. As shown in Fig. 3, insulin can stimulate multiple actions in the vascular cells. We have separated these actions into anti-atherogenic and atherogenic categories. We believe that normally, at physiological conditions without hyperglycemia or insulin resistance, insulin has anti-atherogenic actions. An example of insulin's anti-atherogenic actions is the ability to increase nitrous oxide production, which can cause vasodilatation and retard the migration and growth of arterial smooth muscle cells. The loss of the anti-atherogenic effects of insulin at physiological levels, as in an insulin-resistant state, will by itself enhance the rate of atherosclerosis. However, if hyperinsulinemia also exists, it may also contribute to the atherogenic process by stimulating the growth and production of extracellular matrix, which generally requires much higher concentrations of insulin. One possible molecular explanation by which insulin could lose its metabolic effects but still retain its growth effect is the finding of selective insulin resistance in the signal transduction pathways of insulin in vascular cells. We and others have demonstrated that vascular cells contain a significant number of high-affinity insulin receptors, which are structurally similar to those in other tissues (37). Again, similar to those in other tissues, insulin receptors in the vascular cells can activate at least two different signal transduction pathways of PI3-kinase and mitogen-activated protein (MAP) kinase cascades (40). The activation of the PI3 kinase pathway by insulin requires the tyrosine phosphorylation of insulin receptor substrate (IRS) 1 and 2 proteins. In contrast, the activation of MAP kinase may not require mediation by IRS proteins. It has also been shown by others in nonvascular cells that the activation of the PI3 kinase pathway by insulin mediates metabolic effects, such as glucose transport, whereas the activation of MAP kinase pathways are responsible for chronic effects, such as growth (41). Therefore, we postulate that in insulin-resistant states, the pathway leading from insulin receptors to the activation of PI3 kinase could be blunted in the vascular tissues, whereas insulin's effect on MAP kinase is not affected because of its lack of requirements for IRS docking proteins. In addition, we postulate that the activation PI3 kinase is responsible for most of insulin's anti-atherogenic actions, such as NO production or eNOS gene expression. Thus, in insulin-resistant or insulin-deficient states, insulin's actions through PI3 kinase are lost, which leads to a state of increased risk of atherosclerosis, even without hyperinsulinemia. However, if hyperinsulinemia is present, it may enhance the atherogenic actions of insulin. This hypothesis would explain the increases in cardiovascular disease in both insulin-resistant and insulin-deficient states. This is an important point, as both type 1 and type 2 diabetic patients suffer from increased risk of cardiovascular disease. In addition, the state of hyperglycemia, as we explained earlier, could also inhibit insulin's anti-atherogenic effect, because the activation of PKC- isoform has been reported to inhibit insulin's effect on the activation of the PI3 kinase pathway.

CONCLUSION— These results firmly establish that hyperglycemic or diabetic conditions activate the DAG-PKC signal transduction pathway. The initiating factors are chiefly metabolic, with hyperglycemia as the main element. The finding that secondary metabolic products of glucose, such as glycation products and oxidants, can also increase DAG-PKC suggests that the activation of DAG-PKC could be a common downstream mechanism by which multiple byproducts of glucose exert their adverse effects. It is not surprising that changes in DAG-PKC may serve this role, because this signal transduction pathway is known to regulate many vascular actions and functions. Insulin is also important for vascular functions. The loss of insulin's anti-atherogenic actions alone in insulin-resistant or insulin-deficient states will enhance the atherosclerotic process even in the absence of hyperinsulinemia. Thus, hyperglycemia and loss of insulin's vascular actions are most likely the two most important risk factors specific to the diabetic states that are responsible for the increased risk of cardiovascular pathologies in diabetic patients.

Acknowledgments— These studies were the resulting studies supported by NIH grants EY09178-05, EY05110, and DK53105-01.

The authors would like to express their appreciation to Luisa Dello Iacono for the secretarial assistance in preparing this manuscript.

References
1. The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 329:977–986, 1993

2. Kuwabara T, Cogan DG: Retinal vascular patterns VI: mural cells of the retinal capillaries. Arch Ophthalmol 69:492a–502a, 1963

3. Krowlewski AS, Warram JH, Rand LI, Kahn CR: Epidemiologic approach to etiology of type I diabetes mellitus and its complications. N Engl J Med 329:977–986, 1993

4. Brownlee M: Advanced protein glycosylation in diabetes and aging. Annu Rev Med 46:223–234, 1995

5. King GL, Shiba T, Oliver J, Inoguchi T, Bursell S-E: Cellular and molecular abnormalities in the vascular endothelium of diabetes mellitus. Annu Rev Med 45:179–188, 1994

6. Greene D, Lattimer SA, Sima AAF: Sorbitol, phosphoinositides and sodium-potassium-ATPase in the pathogenesis of diabetic complications. N Engl J Med 316:599–606, 1987

7. Baynes JW: Role of oxidative stress in development of complications in diabetes. Diabetes 40:405–412, 1991

8. Nishizuka Y: Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9:484–496, 1995

9. King GL, Ishii H, Koya D: Diabetic vascular dysfunctions: a model of excessive activation of protein kinase C. Kidney Int 52:S77–S85, 1997

10. Dereubertis FR, Craven PA: Activation of protein kinase C in glomerular cells in diabetes: mechanisms and potential link to the pathogenesis of diabetic glomerulopathy. Diabetes 43:1–8, 1994

11. Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W, King GL: Preferential elevation of protein kinase C isoform II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci U S A 89:11059–11062, 1992

12. Koya D, Jirousek MR, Lin Y-W, Ishii H, Kuboki K, King GL: Characteristics of protein kinase C isoform activation on the gene expression of transforming growth factor , extracellular matrix components and prostanoids in the glomeruli of diabetic rats. JCI 100:115–126, 1997

13. Xia P, Kramer RM, King GL: Identification of the mechanism for the inhibition of NA⁺, K⁺-adenosine triphosphatase by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A₂. J Clin Invest 96:733–740, 1995

14. Yamamoto T, Nakamura T, Noble NA, Ruoslahti E, Border WA: Expression of transforming growth factor is elevated in human and experimental diabetic nephropathy. Proc Natl Acad Sci U S A 90:1814–1818, 1993

15. Sharma K, Jin Y, Guo J, Ziyadeh FN: Neutralization of TGF- by anti-TGF- antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 45:522–530, 1996

16. Ziyadeh FN, Sharma K, Ericksen M, Wolf G: Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-. J Clin Invest 93:536–542, 1994

17. Hostetter TH, Troy JL, Breener BM: Glomerular hemodynamics in experimental diabetes mellitus. Kidney Int 19:410–415, 1981

18. Anderson S, Jung FF, Ingelfinger JR: Renal renin-angiotensin system in diabetes: functional immunohistochemical and molecular biological correlations. Am J Physiol 265:F477–F486, 1993

19. Komers R, Allen TJ, Cooper ME: Role of endothelium-derived nitric oxide in the pathogenesis of the renal hemodynamic changes of experimental diabetes. Diabetes 43:1190–1197, 1994

20. Tesfamariam B, Brown ML, Cohen RA: Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C. J Clin Invest 87:1643–1648, 1991

21. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell S-E, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC inhibitor. Science 272:728–731, 1996

22. Stasek JE, Patterson CE, Garcia JGN: Protein kinase C phosphorylates caldesmon₇₇ and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. J Cell Physiol 153:62–75, 1992

23. Nagpala PG, Malik AB, Vuong PT, Lum H: Protein kinase C ₁ overexpression augments phorbol ester-induced increase in endothelial permeability. J Cell Physiol 166:249–255, 1996

24. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, Pasquale LR, Thieme H, Iwamoto MA, Park JE, Nguyen HV, Aiello LM, Ferrara N, King GL: Vascular endothelial growth factor in ocular fluids of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 331:1480–1487, 1994

25. Xia P, Aiello LP, Ishii H, Jiang Z, Park DJ, Robinson GS, Takagi H, Newsome WP, Jirousek MR, King GL: Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clin Invest 98:2018–2026, 1996

26. Williams B, Gallachen B, Patel H, Orme C: Glucose-induced protein kinase C activation regulates vascular permeability factor mRNA expression and peptide production by vascular smooth muscle cells in vitro. Diabetes 46:1497–1503, 1997

27. Lewis EJ, Hunsicker LG, Bain RP, Rhode RD: The effect of angiotensin converting enzyme inhibitor on diabetic nephropathy. N Engl J Med 329:1456–1462, 1993

28. Kunisaki M, Bursell S-E, Umeda F, Nawata H, King GL: Normalization of diacylglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabetes 43:1372–1377, 1994

29. Kunisaki M, Bursell S-E, Clermont AC, Ishii H, Ballas LM, Jirousek MR, Umeda F, Nawata H, King GL: Vitamin E prevents diabetes-induced abnormal retinal blood flow via the diacylglycerol-protein kinase C pathway. Am J Physiol 269:E239–E246, 1995

30. Tasinato A, Boscoboinik D, Bartoli GM, Maroni P, Azziz A: d--Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition and is independent of its antioxidant properties. Proc Natl Acad Sci U S A 92:12190–12194, 1995

31. Koya D, Lee I-K, Ishii H, Kanoh H, King GL: Prevention of glomerular dysfunctions in diabetic rats by treatment of d--tocopherol. J Am Soc Nephrol 8:426–435, 1997

32. Raman M, Nesto RW: Heart disease in diabetes mellitus. Endocrinol Metab Clin North Am 25:425–438, 1996

33. Wakasaki H, Koya D, Schuen FJ, Jirousek MR, Ways DK, Hoit BD, Walsh RA, King GL: Targeted overexpression of protein kinase C isoform in myocardium causes cardiomyopathy. Proc Natl Acad U S A 94:9320–9325, 1997

34. Lamarche B, Mauriege P, Cantin B, Dagenais GR, Moorjani S, Lupien PJ: Hyperinsulinemia as an independent risk factor for ischemic heart disease. N Engl J Med 334:952–957, 1996

35. Feener EP, King GL: Vascular dysfunction in diabetes mellitus. Lancet 350:SI9–SI13, 1997

36. Colwell JA, Lopes-Virella M, Halushka PV: Pathogenesis of atherosclerosis in diabetes mellitus. Diabetes Care 4:121–133, 1981

37. King GL, Davidheiser S, Banskota N, Oliver J, Inoguchi T: Insulin receptors and actions on vascular cells. In Novo Nordisk Foundation Symposium No. 5. Smith U, Bruun NE, Hedner T, Hokfelt B, Eds. Amsterdam,Excerpta Medica, 1991, p. 183–187

38. Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G, Baron AS: Obesity/insulin resistance is associated with endothelial dysfunction. J Clin Invest 97:2601–2610, 1996

39. Savage PJ, Saad MF: Insulin and atherosclerosis: villain, accomplice or innocent bystander? Br Heart J 69:473–475, 1993

40. Follo F, Kahn CR, Hansen H, Bouchie JL, Feener EP: Angiotensin II inhibits insulin signaling in aortic smooth muscle cells at multiple levels: a potential role for serine phosphorylation in insulin/angiotensin II crosstalk. J Clin Invest 100:2158–2169, 1997

41. Bruning JC, Winnay J, Cheatham B, Kahn CR: Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells. Mol Cell Biol 17:1513–1521, 1997

From the Research Division of Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts.

Address correspondence and reprint requests to George L. King, MD, One Joslin Place, Boston, MA 02215. E-mail: kingg@joslab.harvard.edu.

Received for publication 6 July 1998 and accepted in revised form 12 November 1998.

G.L.K. has received honoraria and research grants from Eli Lilly.

Abbreviations: cPLA₂, cytosolic phospholipase A₂; DAG, diacylglycerol; ECM, extracellular matrix; eNOS, endothelial cell nitric oxide synthase; iNOS, inducible NO synthase; IRS, insulin receptor substrate; MAP, mitogen-activated protein; PI, phosphatidylinositide; PGE₂, prostaglandin E₂; PKC, protein kinase C; TGF-, transforming growth factor-; VEGF, vascular endothelial growth factor; VPF, vascular permeability factor.

A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

This article is based on a presentation at a conference organized by the Indiana University Diabetes Research and Training Center. The conference and the publication of this article were made possible by an unrestricted educational grant from Eli Lilly and Company.

Copyright © 1999 American Diabetes Association
Last updated: 3/99
For ADA Related Issues contact CustomerService@diabetes.org

For Technical Issues contact webmaster@diabetes.org