Endothelial Insulin Resistance Exacerbates Experimental Periodontitis.

Epidemiological studies suggest that the severity of periodontitis is higher in people with diabetes than in healthy individuals. Insulin resistance might play a crucial role in the pathogenesis of multiple diabetic complications and is reportedly induced in the gingiva of rodents with type 2 diabetes; however, the molecular mechanisms underlying the pathogenesis of diabetes-related periodontitis remain unclear. Therefore, we aimed to investigate whether endothelial insulin resistance in the gingiva may contribute to the pathogenesis of periodontitis as well as elucidate its underlying molecular mechanisms. We demonstrated that insulin treatment downregulated lipopolysaccharide (LPS)-induced or tumor necrosis factor α (TNFα)-induced VCAM1 expression in endothelial cells (ECs) via the PI3K/Akt activating pathway, resulting in reduced cellular adhesion between ECs and leukocytes. Hyperglycemia-induced selective insulin resistance in ECs diminished the effect of insulin on LPS- or TNFα-stimulated VCAM1 expression. Vascular endothelial cell-specific insulin receptor knockout (VEIRKO) mice exhibited selective inhibition of the PI3K/Akt pathway in the gingiva and advanced experimental periodontitis-induced alveolar bone loss via upregulation of Vcam1, Tnfα, Mcp-1, Rankl, and neutrophil migration into the gingiva compared with that in the wild-type (WT) mice despite being free from diabetes. We also observed that insulin-mediated activation of FoxO1, a downstream target of Akt, was suppressed in the gingiva of VEIRKO and high-fat diet (HFD)-fed mice, hyperglycemia-treated ECs, and primary ECs from VEIRKO. Further analysis using ECs transfected with intact and mutated FoxO1, with mutations at 3 insulin-mediated phosphorylation sites (T24A, S256D, S316A), suggested that insulin-mediated regulation of VCAM1 expression and cellular adhesion of ECs with leukocytes was attenuated by mutated FoxO1 overexpression. These results suggest that insulin resistance in ECs may contribute to the progression of periodontitis via dysregulated VCAM1 expression and cellular adhesion with leukocytes, resulting from reduced activation of the PI3K/Akt/FoxO1 axis.

Obesity-associated Gingival Vascular Inflammation and Insulin Resistance.

Obesity is a risk factor for periodontitis, but the pathogenic mechanism involved is unclear. We studied the effects of insulin in periodontal tissues during the state of obesity-induced insulin resistance. Gingival samples were collected from fatty (ZF) and lean (ZL, control) Zucker rats. Endothelial nitric oxide synthase (eNOS) expression was decreased, and activities of protein kinase C (PKC) α, ß2, δ, and ϵ isoforms were significantly increased in the gingiva from ZF rats compared with those from ZL rats. Expression of oxidative stress markers (mRNA) and the p65 subunit of NF-κB was significantly increased in ZF rats. Immunohistochemistry revealed that NF-κB activation was also increased in the gingival endothelial cells from transgenic mice overexpressing NF-κB-dependent enhanced green fluorescent protein (GFP) and on a high-fat vs. normal chow diet. Analysis of the gingiva showed that insulin-induced phosphorylation of IRS-1, Akt, and eNOS was significantly decreased in ZF rats, but Erk1/2 activation was not affected. General PKC inhibitor and an anti-oxidant normalized the action of insulin on Akt and eNOS activation in the gingiva from ZF rats. This provided the first documentation of obesity-induced insulin resistance in the gingiva. Analysis of our data suggested that PKC activation and oxidative stress may selectively inhibit insulin-induced Akt and eNOS activation, causing endothelial dysfunction and inflammation.

Effects of solar particle event proton radiation on parameters related to ferret emesis.

The effectiveness of simulated solar particle event (SPE) proton radiation to induce retching and vomiting was evaluated in the ferret experimental animal model. The endpoints measured in the study included: (1) the fraction of animals that retched or vomited, (2) the number of retches or vomits observed, (3) the latency period before the first retch or vomit and (4) the duration between the first and last retching or vomiting events. The results demonstrated that γ ray and proton irradiation delivered at a high dose rate of 0.5 Gy/min induced dose-dependent changes in the endpoints related to retching and vomiting. The minimum radiation doses required to induce statistically significant changes in retching- and vomiting-related endpoints were 0.75 and 1.0 Gy, respectively, and the relative biological effectiveness (RBE) of proton radiation at the high dose rate did not significantly differ from 1. Similar but less consistent and smaller changes in the retching- and vomiting-related endpoints were observed for groups irradiated with γ rays and protons delivered at a low dose rate of 0.5 Gy/h. Since this low dose rate is similar to a radiation dose rate expected during a SPE, these results suggest that the risk of SPE radiation-induced vomiting is low and may reach statistical significance only when the radiation dose reaches 1 Gy or higher.

The role of protein kinase C activation in diabetic nephropathy.

Diabetic nephropathy is the leading cause of end-stage renal disease worldwide and an independent risk factor for all-cause and cardiovascular mortalities in diabetic patients. New insights into the molecular mechanisms that underlie the development and progression of microvascular complications of diabetes including nephropathy are emerging rapidly from experimental and clinical studies. Chronic hyperglycemia is a major initiator of diabetic microvascular complications. Activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway, enhanced polyol pathway, increased oxidative stress, and overproduction of advanced glycation end products have all been proposed as potential cellular mechanisms by which hyperglycemia induces diabetic vascular complications. The DAG-PKC pathway contributes to vascular function in many ways such as the regulation of endothelial permeability, vasoconstriction, extracellular matrix synthesis/turnover, cell growth, angiogenesis, cytokine activation, and leukocyte adhesion. We will briefly review the current knowledge base regarding the pathogenic role for the activation of DAG-PKC pathway in diabetic nephropathy and other microvascular complications of diabetes. The results from animal studies and key clinical studies investigating specific effects of the PKC isoforms on the renal and other vascular tissues to induce diabetic complications are also reviewed.

Angiotensin AT(1) receptor stimulates heat shock protein 27 phosphorylation in vitro and in vivo.

The angiotensin type 1 receptor (AT(1)) exerts a variety of its signaling and cellular actions through its effects on protein phosphorylation. Phosphoproteomic analysis of angiotensin (Ang) II-stimulated aortic smooth muscle cells revealed that heat shock protein 27 (HSP27) represents a major protein phosphorylation target of the AT(1) signaling pathway. Stimulation of cells with Ang II resulted in 1.7-fold (P<0.05) and 5.5-fold (P<0.001) increases in HSP27 phosphoisoforms at pI 5.7 and pI 5.4, respectively. This was accompanied by a 54% (P<0.01) decrease in the nonphosphorylated HSP27 isoform, located at pI 6.4. Treatment of samples with alkaline phosphatase reversed this redistribution of HSP27 phosphoisoforms. Ang II-stimulated HSP27 phosphorylation was completely blocked by pretreatment of cells with the AT(1) antagonist CV11974. Phosphoamino acid analysis demonstrated that Ang II-induced phosphorylation of both HSP27 phosphoisoforms occurred exclusively on serine. Protein kinase C inhibition completely blocked phorbol ester-induced HSP27 phosphorylation but did not impair Ang II-stimulated phosphorylation of HSP27, suggesting that AT(1) increased HSP27 phosphorylation by a protein kinase C-independent pathway. Intrajugular infusion of Ang II in rats increased HSP27 in aorta by 1.7-fold (P<0.02), and this response was inhibited by CV11974. These results suggest that Ang II-induced HSP27 phosphorylation is a physiologically relevant AT(1) signaling event. Because serine phosphorylation of HSP27 blocks its ability to cap F-actin, Ang II/AT(1)-induced HSP27 phosphorylation may play a key role in actin filament remodeling required for smooth muscle cell migration and contraction.

Human vascular smooth muscle cells of diabetic origin exhibit increased proliferation, adhesion, and migration.

PURPOSE: Patients with diabetes mellitus (DM) experience progressive macrovascular atherosclerosis and intimal hyperplastic restenosis with increased frequency as compared with nondiabetic patients. These observations suggest that vascular smooth muscle cells (VSMCs) behave in a phenotypically different and more aggressive manner in diabetic patients. In this study, we compared the in vitro rates of proliferation, adhesion, and migration of human VSMCs obtained from diabetic and nondiabetic patients. METHODS: Human VSMC cultures were isolated from 23 diabetic patients (9 artery, 14 vein) and 15 nondiabetic patients (9 artery, 6 vein) with extensive lower extremity atherosclerosis. All patients were between 61 and 78 years of age (average: 68.4 years [diabetic]; 67.3 years [nondiabetic]). All diabetic patients had type 2 DM. Vascular specimens were obtained at the time of amputation from infragenicular arteries and during arterial revascularization from saphenous veins. Cells from passages 2 and 3 were assayed for their proliferative capacity with total DNA fluorescence photometry and for adhesion and migration with a modified Boyden chamber. RESULTS: The average duration of diabetes was 11.6 +/- 4.1 years. The average number of diabetic complications (retinopathy, neuropathy, nephropathy, coronary artery disease) was 2.8 +/- 0.7 per patient. Diabetic VSMCs exhibited abnormal morphology in cell culture with loss of the normal hill and valley configuration. Proliferation was significantly increased in VSMCs of diabetic origin (156 +/- 57 absorption units) as compared with those of nondiabetic origin (116 +/- 42 absorption units) (P <.001). Diabetic VSMCs demonstrated significantly greater adhesion (63.6 +/- 24 per high-power field vs 37.9 +/- 13 per high-power field; P =.002) and migration (397 +/- 151 per low-power field vs 121 +/- 99 per low-power field; P =.001) rates. CONCLUSIONS: Diabetic VSMCs exhibit significantly increased rates of proliferation, adhesion, and migration as well as abnormal cell culture morphology suggestive of abnormal contact inhibition. These observations of human VSMCs in culture are consistent with the increased rate of infragenicular atherosclerosis and the increased rates of restenosis observed clinically in diabetic patients. The atherosclerosis- and intimal hyperplasia-promoting behavior exhibited appears to be intrinsic to the DM-VSMC phenotype and must be considered when designing methods to limit atherosclerosis and intimal hyperplasia in diabetic patients.

Protein kinase C and the development of diabetic vascular complications.

Hyperglycemic control in diabetes is key to preventing the development and progression of vascular complications such as retinopathy, nephropathy and neuropathy. Increased activation of the diacylglycerol (DAG)-protein kinase C (PKC) signal transduction pathway has been identified in vascular tissues from diabetic animals, and in vascular cells exposed to elevated glucose. Vascular abnormalities associated with glucose-induced PKC activation leading to increased synthesis of DAG include altered vascular blood flow, extracellular matrix deposition, basement membrane thickening, increased permeability and neovascularization. Preferential activation of the PKCbeta isoform by elevated glucose is reported to occur in a variety of vascular tissues. This has lead to the development of LY333531, a PKCbeta isoform specific inhibitor, which has shown potential in animal models to be an orally effective and nontoxic therapy able to produce significant improvements in diabetic retinopathy, nephropathy, neuropathy and cardiac dysfunction. Additionally, the antioxidant vitamin E has been identified as an inhibitor of the DAG-PKC pathway, and shows promise in reducing vascular complications in animal models of diabetes. Given the overwhelming evidence indicating a role for PKC activation in contributing to the development of diabetic vascular complications, pharmacological therapies that can modulate this pathway, particularly with PKC isoform selectivity, show great promise for treatment of vascular complications, even in the presence of hyperglycemia.

Endothelial dysfunction in diabetes mellitus: role in cardiovascular disease.

Diabetes mellitus is associated with an increased risk of cardiovascular disease (CVD), even in the presence of intensive glycemic control. Substantial clinical and experimental evidence suggests that both diabetes and insulin resistance cause a combination of endothelial dysfunctions, which may diminish the anti-atherogenic role of the vascular endothelium. Endothelial dysfunctions that have been described include decreased endothelium-dependent vasorelaxation, increased leukocyte-endothelial cell adhesion and vascular permeability, and the altered production of a variety of vasoactive substances, which affect coagulation, extracellular matrix homeostasis, and smooth muscle physiology. The primary mechanisms that contribute to these endothelial dysfunctions in diabetes appear to involve the activation of protein kinase C (PKC) pathways, increased non-enzymatic glycation, increased oxidant stress, and reduced endothelial insulin action. In addition, many of the adverse effects of these abnormalities associated with hyperglycemia and insulin resistance are mediated and amplified by potent vasoactive hormones including angiotensin II, transforming growth factor-beta, and vascular endothelial growth factor. Multiple interventions have been shown to improve endothelial dysfunction in diabetes, including PKC inhibition, infusion of soluble receptors for advanced glycation end-products, antioxidant and insulin supplementation, and angiotensin-converting enzyme inhibition. These findings are consistent with a model involving a combination of factors contributing to the etiology of the endothelial dysfunctions in diabetes. Further work is needed to determine whether endothelial function can be used as a therapeutic target to reduce CVD and improve clinical outcomes.

Performance decrement after combined exposure to ionizing radiation and Shigella sonnei.

Ionizing radiation could increase morbidity from common bacterial infections in military personnel on the modern battlefield. The combined effects of a sublethal dose of ionizing radiation and the bacterial diarrheal agent Shigella sonnei on body weight and forelimb grip strength in mice were assessed over a 30-day period. Individually housed B6D2F1 female mice were divided into four groups: control, sham irradiation + gavage with saline vehicle; 3 Gy 60Co gamma radiation at 0.4 Gy/min radiation + saline gavage; sham irradiation + 1.3 x 10(8) colony-forming units (CFUs) S. sonnei via gavage, administered 4 days postirradiation; and the combination of 3 Gy 60Co gamma radiation + 1.3 x 10(8) CFUs S. sonnei. Behavioral tests were conducted 3 days preirradiation and on days 9, 14, and 22 postirradiation. Body weight was significantly reduced in the radiation + Shigella group on days 5 to 10 postirradiation. Forelimb grip strength was reduced for mice in the radiation + Shigella group on days 9 and 14 postirradiation. These data demonstrate that an exposure to gamma radiation in combination with the bacterial agent S. sonnei can lead to a synergistic loss of body weight and degradation in performance.

Protein kinase C activation and its pharmacological inhibition in vascular disease.

Vascular complications in diabetes mellitus are known to be associated with the activation of the protein kinase C (PKC) pathway through the de novo synthesis of diacylglycerol (DAG) from glycolytic intermediates. Specific PKC isoforms, mainly the beta- and delta-isoforms, have been shown to be persistently activated in diabetic mellitus. Multiple studies have reported that the activation of PKC leads to increased production of extracellular matrix and cytokines, enhances contractility, permeability and vascular cell proliferation, induces the activation of cytosolic phospholipase A2 and inhibits the activity of Na+-K+-ATPase. These events are not only frequently observed in diabetes mellitus but are also involved in the actions of vasoactive agents or oxidative stress. Inhibition of PKC by two different kinds of PKC inhibitors - LY333531, a selective PKC-beta-isoform inhibitor, and vitamin E, d-alpha-tocopheron - were able to prevent or reverse the various vascular dysfunctions in vitro and in vivo. Clinical studies using these compounds are now ongoing to evaluate the significance of DAG-PKC pathway activation in the development of vascular complications in diabetic patients.

von Willebrand factor and retinal circulation in early-stage retinopathy of type 1 diabetes.

OBJECTIVE: Although retinopathy is a common microvascular complication of type 1 diabetes, the mechanism for this complication is still unknown. Changes in retinal circulation have been noted before the development of overt retinal pathology. Because von Willebrand factor (vWF) is a marker for endothelial dysfunction and mediates platelet adhesion, we determined if there was an association between vWF and retinal circulation in the early stages of diabetic retinopathy. RESEARCH DESIGN AND METHODS: Twenty subjects (aged 32.4 +/- 7.8 years) with type 1 diabetes and minimal or no retinopathy were studied. The mean duration of diabetes was 4.7 +/- 2.6 years. Data were collected at baseline and after 4 months of 1,800 IU vitamin E therapy or placebo. Retinal circulation was evaluated by video fluorescein angiography. Plasma vWF antigen levels were measured by enzyme-linked immunosorbent assay and fibrinogen by the Clauss method. RESULTS: Retinal blood flow was negatively correlated with vWF levels (r = -0.44, P = 0.008), whereas retinal circulation time was positively correlated with vWF levels (r = 0.33, P = 0.048). Fibrinogen levels were not significantly associated with either retinal index. However, fibrinogen levels were positively associated with HbA1c levels (r = 0.34, P = 0.01), indicating an association between poor glycemic control and higher fibrinogen levels. CONCLUSIONS: Increased vWF was associated with a prolonged retinal circulation time and reduced retinal blood flow in early-stage retinopathy of type 1 diabetes. Reduced blood flow associated with increased vWF levels may promote stasis in the retinal circulation and lead to local hypoxemia. These changes might contribute to the microvascular complications of diabetes. Whether the vWF levels predict retinal complications deserves further investigation.

Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells.

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.

Endothelin-3 regulation of retinal hemodynamics in nondiabetic and diabetic rats.

PURPOSE: To investigate the mechanisms of action of endothelin (ET)-3 on the regulation of retinal hemodynamics in diabetic and nondiabetic rats. METHODS: Retinal blood flow changes were measured using video fluorescein angiography. Measurements were made before and after intravitreal injections of different ET-3 concentrations in nondiabetic rats and rats with streptozotocin (STZ)-induced diabetes. The effect of ET-3 on retinal blood flow was also investigated in nondiabetic rats after pretreatment with N:(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor; BQ-788, an ET receptor B (ETB) antagonist; and BQ-123, an ET receptor A (ETA) antagonist. Control animals were injected intravitreally with vehicle alone. RESULTS: In nondiabetic rats, ET-3 induced a dose-dependent rapid increase in retinal blood flow 2 minutes after intravitreal injection (maximal at 10(-)(8) M, P < 0. 01) followed 15 and 30 minutes after ET-3 injection by dose-dependent decreases in retinal blood flow (maximal effect at 10(-)(6) M, P < 0.05). The ET-3-stimulated retinal blood flow increase was inhibited by 10(-)(4) M BQ-788 (P < 0.01) and 10(-)(3) M L-NMMA (P < 0.05). The ET-3-stimulated decrease in retinal blood flow at later times (15 minutes) was inhibited (P < 0.03) by 10(-4) M BQ-123. In diabetic rats, baseline retinal blood flows were decreased compared with nondiabetic rats (P < 0.01), showed dose-dependent increases 2 minutes after ET-3 injection (P < 0.03), and at later times remained significantly increased (P < 0.05) in contrast to flows in nondiabetic rats. CONCLUSIONS: The ET-3-induced initial rapid retinal blood flow increase in nondiabetic rats is mediated by the ET-3/ETB and NOS action. The subsequent retinal blood flow decrease is mediated by ET-3/ETA action. Diabetic rats showed comparable ET-3-induced retinal blood flow increases indicating normal ET-3/ETB action. However, at later times, retinal blood flow remained increased, suggesting an abnormal ET-3/ETA action.

Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation.

Enhanced actions or levels of endothelin-1 (ET-1), a potent vasoconstrictor, have been associated with decreased blood flow in the retina and peripheral nerves of diabetic animals and may be related to the development of pathologies in these tissues. Hyperglycemia has been postulated to increase ET-1 secretion in endothelial cells. We have characterized the mechanism by which elevation of glucose is increasing ET-1 mRNA expression in capillary bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRPC). Elevation of glucose, but not mannitol, from 5.5 to 25 mmol/l for 3 days increased membranous protein kinase C (PKC) activities and ET-1 mRNA in parallel levels by 2-fold in BREC and BRPC. These effects were reversed by decreasing glucose levels to 5.5 mmol/l for an additional 2 days. Glucose-induced ET-1 overexpression was inhibited by a general PKC inhibitor, GF109203X, and a mitogen-activated protein kinase kinase inhibitor, PD98059, but not by wortmannin, a phosphatidylinositol 3-kinase inhibitor. By immunoblot analysis, PKC-beta2 and -delta isoforms in BREC were significantly increased relative to other isoforms in the membranous fractions when glucose level was increased. Overexpression of PKC-beta1 and -delta isoforms but not PKC-zeta isoform by adenovirus vectors containing the respective cDNA enhanced in parallel PKC activities, proteins, and basal and glucose-induced ET-1 mRNA expression by at least 2-fold. These results showed that enhanced ET-1 expression induced by hyperglycemia in diabetes is partly due to activation of PKC-beta and -delta isoforms, suggesting that inhibition of these PKC isoforms may prevent early changes in diabetic retinopathy and neuropathy.

Identification of PKC-isoform-specific biological actions using pharmacological approaches.

The protein kinase C (PKC) family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. To date, identification of the physiological function of individual PKC isoforms has been restricted by the availability of few agents that inhibit or activate the isoforms with specificity. More recent approaches that are used to modulate PKC isoforms include oligonucleotide antisense technology, and peptide fragments to either inhibit or promote translocation of PKC isoforms to specific anchoring proteins. In this review, several currently available inhibitors and activators of PKC that display varying degrees of selectivity for the PKC isoforms will be discussed.

Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.

Activation of protein kinase C (PKC) is implicated as an important mechanism by which diabetes causes vascular complications. We have recently shown that a PKC beta inhibitor ameliorates not only early diabetes-induced glomerular dysfunction such as glomerular hyperfiltration and albuminuria, but also overexpression of glomerular mRNA for transforming growth factor beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in streptozotocin-induced diabetic rats, a model for type 1 diabetes. In this study, we examined the long-term effects of a PKC beta inhibitor on glomerular histology as well as on biochemical and functional abnormalities in glomeruli of db/db mice, a model for type 2 diabetes. Administration of a PKC beta inhibitor reduced urinary albumin excretion rates and inhibited glomerular PKC activation in diabetic db/db mice. Administration of a PKC beta inhibitor also prevented the mesangial expansion observed in diabetic db/db mice, possibly through attenuation of glomerular expression of TGF-beta and ECM proteins such as fibronectin and type IV collagen. These findings provide the first in vivo evidence that the long-term inhibition of PKC activation in the renal glomeruli can ameliorate glomerular pathologies in diabetic state, and thus suggest that a PKC beta inhibitor might be an useful therapeutic strategy for the treatment of diabetic nephropathy.

Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo : a specific vascular action of insulin.

BACKGROUND: The vasodilatory effect of insulin can be acute or increase with time from 1 to 7 hours, suggesting that insulin may enhance the expression of endothelial nitric oxide synthase (eNOS) in endothelial cells. The objective of the present study was to characterize the extent and signaling pathways by which insulin regulates the expression of eNOS in endothelial cells and vascular tissues. METHODS AND RESULTS: Physiological concentrations of insulin (10(-10) to 10(-7) mmol/L) increased the levels of eNOS mRNA, protein, and activity by 2-fold after 2 to 8 hours of incubation in cultured bovine aortic endothelial cells. Insulin enhanced eNOS gene expression in microvessels isolated from Zucker lean rats but not from insulin-resistant Zucker fatty rats. Inhibitors of phosphatidylinositol-3 kinase (PI-3 kinase) decreased the effect of insulin on eNOS gene expression, but a general protein kinase C (PKC) inhibitor, GF109203X or PKCbeta isoform inhibitor, LY333531 enhanced eNOS expression. In contrast, PKC activators inhibited both the activation by insulin of PI-3 kinase and eNOS mRNA levels. Overexpression of PKCbeta isoform in endothelial cells inhibited the stimulation by insulin of eNOS expression and PI-3 kinase activities in parallel. CONCLUSIONS: Insulin can regulate the expression of eNOS gene, mediated by the activation of PI-3 kinase, in endothelial cells and microvessels. Thus, insulin may chronically modulate vascular tone. The activation of PKC in the vascular tissues as in insulin resistance and diabetes may inhibit PI-3 kinase activity and eNOS expression and may lead to endothelial dysfunctions in these pathological states.