Activation of the Dagpkc Pathway

One major advance in the understanding of diabetic vascular disease is the unraveling of changes in signal transduction pathways in diabetic states. One of the best-characterized signaling changes is the activation of DAG-PKC pathway. Such activation appears to be related to elevation of DAG, a physiological activator of PKC. Increases in total DAG contents have been demonstrated in a variety of tissues associated with diabetic vascular complications, including retina (78), aorta, heart (79), renal glomeruli (80), and nonvascular tissues as in the liver (81), but not in the brain and peripheral nerves of diabetic animals and patients (Table 4). Increasing glucose levels from 5 to 22 mol/L in the media elevated the cellular DAG contents in aortic endothelial cells and SMCs (79), retinal endothelial cells (78), and renal mesangial cells (82,83). The increase in DAG-PKC reaches maximum in 3-5 days after elevating glucose levels and remain chronically elevated for many years. In fact, we have already shown 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 (79). These data suggest that the activation of DAG-PKC could be sustained chronically and is difficult to reverse, similar to pathways of diabetic complications.

DAG can be generated from multiple pathways. Agonist-induced formation of DAG depends mainly on hydrolysis of phosphatidylinositol by phospholipase C (84). However, this mechanism is most likely minimally involved in diabetes, because inositol phosphate products were not found to be increased by hyperglycemia in aortic cells and glomerular mesangial cells (85,86). When the fatty acids in DAG were analyzed (87), DAG induced by high-glucose condition has predominantly palpitate- and oleic- acid-enriched composition, whereas DAG generated from hydrolysis of phosphatidylinositol has the composition of 1-stearoly-2-arachidonyl-,SW-glycerol (88). In labeling studies using [6-3H]- or [U-14C]- glucose, we have shown that elevated glucose increase the incorporation of glucose into the glycerol backbone of DAG in aortic endothelial cells (87), aortic SMCs (89), and renal glomeruli (90). These facts indicate that the increased DAG levels in high-glucose condition are mainly derived from the de novo pathway (Fig. 2).

It is also possible that DAG is produced through the metabolism of phosphatidylcholine as a result of the activation of phospholipase D (91). One potential pathway for the increase in DAG is the result of glyco-oxidation inducing activation of the DAG pathway because oxidants such as H2O2 are known to activate DAG-PKC pathway (Fig. 3) (92). We have reported that 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 (74). We have confirmed that vitamin E can inhibit PKC activation probably by decreasing DAG levels rather than inhibiting PKC, because the direct addition of vitamin E to purified PKC-a or -P isoforms in vitro has no inhibitory effect (93).

PKC belongs to a family of serine-threonine kinases and plays a key role in intracellular signal transduction for hormones and cytokines. There are at least 11 isoforms of PKC and are classified as conventional PKCs (a, P1, P2, y); novel PKCs (8, £, 0, and atypical PKCs (£,X) (94,95). Multiple isoforms of PKC including a, P1, P2,5, e, and C, are all expressed in endothelial cells (79,96). Activation of PKC has been suggested to play key roles in the development of diabetic cardiovascular complications (97).

The activation of PKC by hyperglycemia appears to be tissue-selective, because it has been noted in the retina, aorta, heart, and glomeruli but not in the brain and peripheral nerves in diabetic animals (Table 4). Among the various PKC isoforms, PKC-P and -8 appear to be preferentially activated in the aorta and heart of diabetic rats (79) and in cultured aortic SMCs exposed to high levels of glucose (74). However, increases in multiple PKC isoforms were observed in some vascular tissues, such as PKC-a, -P2, and -£ in the retina and PKC-a, P1, and 8 in the glomeruli in the glomeruli of diabetic rats (98). Recently, we and others have shown that a number of in vivo abnormalities such as renal mesangial expansion, basement membrane thickening, blood flow, and monocyte activation in diabetic rats can be prevented or normalized using an orally effective specific inhibitor for PKC-P isoform LY333531 (90). One of the early changes in the vasculature in diabetic states is the reduced bioavailability of endothelium-derived NO, which further aggravates endothelial dysfunctions. This process is apparently at least partly caused by the activation of PKC-P by hyperglycemia. Beckman and colleagues applied forearm hyperglycemic clamps on fourteen healthy subjects to mimic the effects and demonstrated that endothelium-dependent vasodilation in response to methacholine chloride is decreased in hyperglycemia as compared to that in euglycemic conditions (99). The reduction of vasodilation can be normalized by oral treatment of PKC-P-selective inhibi-

Pkc Insulin Resistance

Fig. 3. Schematic diagram of pathways utilized by hyperglycemia to induce pathological changes in the vasculature. Hyperglycemia stimulates de novo synthesis of DAG that further activates multiple isoforms of PKC. Activation of the a, p, and 8 isoforms have all been reported. This will in turn affect the activity of other intracellular signaling pathways such as the Ras/MEK/MAPK, p38 MAPK and PI3K/Akt pathways. Alteration of key enzymes determining cellular homeostasis, i.e., NADPH oxidase, Na+/K+-ATPase; eNOS, COR 2 transferase has also been documented. All these changes can have profound impact on the regulation of vascular cell biology including cell cycle progression, gene expression, endothelial cell dysfunctions and hemodynamic change that constitute the cellular basis of diabetic vascular complications. PLC; phospholipase D, PLC; Phospholipase C, eNOS; endothelial nitric oxide synthase, Rb, retinoblastoma; Egr-1, early growth response-1, GSK-3p; Glycogen synthase kinase-3p, IKKa; Ik B kinasea, VEGF; vascular endothelial growth factor, ANP; atrial natriuretic peptide; PDGF, platelet-derived growth factor; ET-1; endothelin-1, CTGF, connective tissue growth factor; TGF-p, transforming growth factor-p; ICAM, intercellular adhesion molecules; SOCS2, suppressor of cytokine signaling.

Fig. 3. Schematic diagram of pathways utilized by hyperglycemia to induce pathological changes in the vasculature. Hyperglycemia stimulates de novo synthesis of DAG that further activates multiple isoforms of PKC. Activation of the a, p, and 8 isoforms have all been reported. This will in turn affect the activity of other intracellular signaling pathways such as the Ras/MEK/MAPK, p38 MAPK and PI3K/Akt pathways. Alteration of key enzymes determining cellular homeostasis, i.e., NADPH oxidase, Na+/K+-ATPase; eNOS, COR 2 transferase has also been documented. All these changes can have profound impact on the regulation of vascular cell biology including cell cycle progression, gene expression, endothelial cell dysfunctions and hemodynamic change that constitute the cellular basis of diabetic vascular complications. PLC; phospholipase D, PLC; Phospholipase C, eNOS; endothelial nitric oxide synthase, Rb, retinoblastoma; Egr-1, early growth response-1, GSK-3p; Glycogen synthase kinase-3p, IKKa; Ik B kinasea, VEGF; vascular endothelial growth factor, ANP; atrial natriuretic peptide; PDGF, platelet-derived growth factor; ET-1; endothelin-1, CTGF, connective tissue growth factor; TGF-p, transforming growth factor-p; ICAM, intercellular adhesion molecules; SOCS2, suppressor of cytokine signaling.

tor LY333531 (32 mg per day) (99). These data support that the activation of PKC-p isoform is involved in the development of some aspects of diabetic vascular complications.

For a hyperglycemia-induced change to be credible as a causal factor of diabetic complications, it has to be shown to be chronically altered, to be difficult to reverse, to cause similar vascular changes when activated without diabetes, and to be able to prevent complications when it is inhibited. So far, we have presented evidence on the DAG-PKC activation that fulfills at least three of these criteria. Clinical studies using a PKC-P inhibitor are now in a phase II/III clinical trial to determine its usefulness in diabetic retinopathy (100) and neuropathy (101).

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