Insulin Resistance In Endothelial Cells Research Articles

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.

Endothelial Cell Insulin Signaling Regulates CXCR4 (C-X-C Motif Chemokine Receptor 4) and Limits Leukocyte Adhesion to Endothelium.

An activated, proinflammatory endothelium is a key feature in the development of complications of obesity and type 2 diabetes and can be caused by insulin resistance in endothelial cells. We analyzed primary human endothelial cells by RNA sequencing to discover novel insulin-regulated genes and used endothelial cell culture and animal models to characterize signaling through CXCR4 (C-X-C motif chemokine receptor 4) in endothelial cells. CXCR4 was one of the genes most potently regulated by insulin, and this was mediated by PI3K (phosphatidylinositol 3-kinase), likely through FoxO1, which bound to the CXCR4 promoter. CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared with lean controls and 37% higher in db/db mice than db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. SDF-1 (stromal cell-derived factor-1)-the ligand for CXCR4-increased leukocyte adhesion to cultured endothelial cells. This effect was lost after deletion of CXCR4 by gene editing while 80% of the increase was prevented by treatment of endothelial cells with insulin. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling after intravenous injection of SDF-1, but most of this response was prevented in transgenic mice with endothelial overexpression of IRS-1 (insulin receptor substrate-1). Endothelial cell insulin signaling limits leukocyte/endothelial cell interaction induced by SDF-1 through downregulation of CXCR4. Improving insulin signaling in endothelial cells or inhibiting endothelial CXCR4 may reduce immune cell recruitment to the vascular wall or tissue parenchyma in insulin resistance and thereby help prevent several vascular complications.

The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

Endothelial cell insulin resistance contributes to the development of vascular complications in diabetes. Hypoxia-inducible factors (HIFs) modulate insulin sensitivity, and we have previously shown that a negative regulator of HIF activity, CREB-binding protein/p300 (CBP/p300) interacting transactivator-2 (CITED2), is increased in the vasculature of people with type 2 diabetes. Therefore, we examined whether CITED2 regulates endothelial insulin sensitivity. In endothelial cells isolated from mice with a "floxed" mutation in the Cited2 gene, loss of CITED2 markedly enhanced insulin-stimulated Akt phosphorylation without altering extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation. Similarly, insulin-stimulated Akt phosphorylation was increased in aortas of mice with endothelial-specific deletion of CITED2. Consistent with these observations, loss of CITED2 in endothelial cells increased insulin-stimulated endothelial nitric oxide synthase phosphorylation, Vegfa expression, and cell proliferation. Endothelial cells lacking CITED2 exhibited an increase in insulin receptor substrate (IRS)-2 protein, a key mediator of the insulin signaling cascade, whereas IRS-1 was unchanged. Conversely, overexpression of CITED2 in endothelial cells decreased IRS-2 protein by 55% without altering IRS-1, resulting in impaired insulin-stimulated Akt phosphorylation and Vegfa expression. Overexpression of HIF-2α significantly increased activity of the Irs2 promoter, and coexpression of CITED2 abolished this increase. Moreover, chromatin immunoprecipitation (ChIP) showed that loss of CITED2 increased occupancy of p300, a key component of the HIF transcriptional complex, on the Irs2 promoter. Together, these results show that CITED2 selectively inhibits endothelial insulin signaling and action through the phosphoinositide 3-kinase (PI3K)/Akt pathway via repression of HIF-dependent IRS-2 expression. CITED2 is thus a promising target to improve endothelial insulin sensitivity and prevent the vascular complications of diabetes.NEW & NOTEWORTHY Endothelial cell insulin resistance is a major contributor to the development of diabetic complications. In this study, we have shown that CITED2, a transcriptional coregulator, inhibits endothelial insulin signaling through the PI3K/Akt pathway via repression of HIF-dependent IRS-2 expression, and that deletion of CITED2 enhances insulin signaling. Thus, CITED2 represents a novel and promising target to improve insulin sensitivity in endothelial cells and prevent vascular complications in diabetes.

273-LB: Leukocyte-Endothelial Interaction Is Reduced by Insulin-Induced Downregulation of Endothelial CXCR4

An activated, pro-inflammatory endothelium is a key feature of obesity and type 2 diabetes and may contribute to adipose tissue inflammation, atherosclerosis and tumor formation. We have previously published a striking 4-fold increase in leukocyte adhesion to endothelium in mice with endothelial cell knockout of the insulin receptor. Here, we searched for insulin-regulated genes involved in leukocyte-endothelial cell interaction by performing RNA sequencing in primary human endothelial cells. Among genes with the largest change in expression after insulin treatment was C-X-C motif chemokine receptor 4 (CXCR4), which was downregulated by 54%. This reduction was completely prevented by pretreatment with wortmannin but not by U0126, an inhibitor of MAPK signaling. In CD31+ cells isolated by fluorescence-activated cell sorting of enzymatically dissociated tissue, CXCR4 mRNA in was 77% higher in mice with diet-induced obesity compared to lean controls and 37% higher in db/db mice compared to db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. Stromal-derived factor-1 (SDF-1), the ligand for CXCR4, increased leukocyte adhesion to cultured endothelial cells. Insulin treatment of endothelial cells prevented 80% of this increase which in turn was abrogated by plerixafor, a clinically approved CXCR4 antagonist, or deletion of CXCR4 by gene editing. To study gain of endothelial cell insulin signaling in vivo, we examined transgenic mice with endothelial overexpression of insulin receptor substrate-1 (IRS-1). In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling and adhesion after intravenous injection of SDF-1 in wildtype mice which was reduced by 83% in transgenic mice. We conclude that endothelial cell insulin signaling limits leukocyte-endothelial cell interaction through downregulation of CXCR4 and that targeting endothelial CXCR4 may protect against inflammation in insulin resistant states. Disclosure B. Kunkemoeller: None. T. Rathjen: Employee; Self; Bayer AG. J.C. Patti: None. Q. Li: None. K. Park: None. G.L. King: Research Support; Self; Sanofi. C. Rask-Madsen: None.

Acid Sphingomyelinase Down-regulation Alleviates Vascular Endothelial Insulin Resistance in Diabetic Rats.

Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in patients with type 2 diabetes. Acid sphingomyelinase (ASM) is a soluble glycoprotein which plays a vital role in the development and progression of various diseases such as cardiovascular and metabolic diseases. However, it remains unknown if ASM regulates insulin resistance in vascular endothelial cells in type 2 diabetes. ASM down-regulation with gene silencing and selective inhibitor amitriptyline was used in the rat aortic endothelial cells (RAECs) treated with palmitic acid (PA), a common saturated free fatty acid, which is thought to be the major cause of insulin resistance. It was shown that ASM down-regulation increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of pIRS-1-ser307 and AKT-ser473 via ceramide, consequently resulting in the decrease of the production of endothelial nitric oxide synthase (eNOS) and nitric oxide in PA-induced RAECs. We further found that ASM down-regulation blocked the Nox2- and Nox4-dependent superoxide (O2 -· ) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vivo, amitriptyline relieved the vasodilatory response to acetylcholine and restored the level of ceramide, Nox2 and Nox4 in the aorta endothelium of high-fat diet-fed rats following an injection of streptozotocin. Taken together, these results suggest that ASM down-regulation can improve endothelial insulin resistance which is attributed to inhibiting redox signalling in RAECs. Thus, these data support the idea that ASM is a promising clinical biomarker and potential therapeutic target for diabetic vascular complication.

Insulin Action on Endothelial Cells Limits Leukocyte-Endothelial Interaction through CXCR4 and Counteracts Intestinal Tumor Formation in Obesity

An activated, pro-inflammatory endothelium is a key feature of obesity and type 2 diabetes but little is known about its contribution to the increased cancer risk in these conditions. We recently published that insulin resistance in endothelial cells increases VCAM-1 expression, increases recruitment of tumor-associated neutrophils and promotes intestinal tumor formation. Here, we report that tumor-prone Apc(Min/+) mice have 2.1-fold more intestinal tumors during high-fat feeding. However, gain of insulin signaling specifically in endothelial cells by overexpression of IRS1 prevented 44% of tumors in diet-induced obesity. RNA sequencing in primary human endothelial cells identified 17 genes changed by insulin treatment with a log(2) change of at least ±0.5, among them C-X-C motif chemokine receptor 4 (CXCR4), which was downregulated by 54%. Downregulation of CXCR4 after insulin treatment was completely prevented by pretreatment with wortmannin but not by U0126, an inhibitor of MAPK signaling. Using FACS of enzymatically dissociated tissue, CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared to lean controls and 37% higher in db/db mice compared to db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. CXCL12, the ligand for CXCR4, increased leukocyte adhesion to cultured endothelial cells and this was completely prevented by plerixafor, a clinically approved CXCR4 antagonist. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling and adhesion after intravenous injection of CXCL12 and this change was abrogated in transgenic mice with endothelial overexpression of IRS1. We conclude that endothelial cell insulin signaling limit leukocyte-endothelial cell interaction through downregulation of CXCR4 and that improving insulin signaling in endothelial cells may protect against tumor development in obesity. Disclosure T. Rathjen: Employee; Self; Novo Nordisk A/S, Bayer AG. Q. Li: None. K. Park: None. G. Povlsen: Employee; Self; Novo Nordisk A/S. G.S. Olsen: Employee; Self; Novo Nordisk A/S. G.L. King: Research Support; Self; Sanofi-Aventis. C. Rask-Madsen: Research Support; Self; Novo Nordisk A/S.

Argirein alleviates vascular endothelial insulin resistance through suppressing the activation of Nox4-dependent O2- production in diabetic rats.

Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in type 2 diabetes mellitus (T2DM). Therefore, there are great potential clinical implications in developing pharmacological interventions targeting endothelial insulin resistance. Our previous studies indicated that argirein which was developed by combining rhein with L-arginine by a hydrogen bond, could substantially relieved stress related exacerbation of cardiac failure and alleviated cardiac dysfunction in T2DM, which was associated with suppressing NADPH oxidase activity. However, it is unclear whether argirein treatment attenuates the vascular lesion and dysfunction in T2DM and its underlying mechanisms. The rat aortic endothelial cells (RAECs) were used to treat with palmitic acid (PA), a most common saturated free fatty acid, which could induce insulin resistance. It was showed that argirein increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of IRS-1-ser307 and AKT-ser473, consequently resulting in the increase of the production of eNOS and NO in PA-induced RAECs. We further found that argirein blocked the Nox4-dependent superoxide (O2-.) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vitro, argirein increased the release of endothelial NO to relieve the vasodilatory response to acetylcholine and insulin, and restored the expression of Nox4 and pIRS-1-ser307 in the aorta endothelium of high-fat diet (HFD)-fed rats following an injection of streptozocin (STZ). These results suggested that argirein could improve endothelial insulin resistance which was attributed to inhibiting Nox4-dependent redox signaling in RAECs. These studies thus revealed the novel effect of argirein to prevent the vascular complication in T2DM.

ATP2B1 gene Silencing Increases Insulin Sensitivity through Facilitating Akt Activation via the Ca2+/calmodulin Signaling Pathway and Ca2+-associated eNOS Activation in Endothelial Cells.

Endothelial cell insulin resistance may be partially responsible for the higher risk of atherosclerosis and cardiovascular disease in populations with insulin resistance and type 2 diabetes mellitus (T2DM). A genome-wide association study revealed a significant association between the ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) gene and T2DM in two community-based cohorts from the Korea Association Resource Project. However, little is known about the implication of the ATP2B1 gene on T2DM. In the present study, we investigated the role of the ATP2B1 gene in endothelial cell insulin sensitivity. ATP2B1 gene silencing resulted in enhanced intracellular calcium concentrations and increased insulin-induced Akt activation compared to that in the negative siRNA-transfected HUVECs (Human Umbilical Vein Endothelial Cells). The elevated insulin sensitivity mediated by ATP2B1 gene silencing was Ca2+/calmodulin-dependent, as verified by administration of the calcium chelator BAPTA-AM or the calmodulin-specific antagonist W7. Moreover, higher levels of phosphorylation of eNOS (Ser1177) were observed in ATP2B1-silenced HUVECs. In addition to BAPTA-AM and W7, L-NAME, an eNOS antagonist, abolished insulin-induced Akt phosphorylation at Ser473 in both si-Neg and si-ATP2B1-transfected endothelial cells. These results indicate that the enhanced insulin sensitivity in ATP2B1-silenced endothelial cells is alternatively dependent on an increase in intracellular Ca2+ and the subsequent activation of the Ca2+/calmodulin/eNOS/Akt signaling pathway. In summary, ATP2B1 gene silencing increased insulin sensitivity in endothelial cells by directly modulating the Ca2+/calmodulin signaling pathway and via the Ca2+/calmodulin/eNOS/Akt signaling pathway alternatively.

The role of miR-190a in methylglyoxal-induced insulin resistance in endothelial cells

Methylglyoxal (MGO) is a reactive dicarbonyl produced as by-product of glycolysis, and its formation is heightened in hyperglycaemia. MGO plasma levels are two-fold to five-fold increased in diabetics and its accumulation promotes the progression of vascular complications. Impairment of endothelium-derived nitric oxide represents a common feature of endothelial dysfunction in diabetics. We previously demonstrated that MGO induces endothelial insulin resistance. Increasing evidence shows that high glucose and MGO modify vascular expression of several microRNAs (miRNAs), suggesting their potential role in the impairment of endothelial insulin sensitivity. The aim of the study is to investigate whether miRNAs may be involved in MGO-induced endothelial insulin resistance in endothelial cells.MGO reduces the expression of miR-190a both in mouse aortic endothelial cells (MAECs) and in aortae from mice knocked-down for glyoxalase-1. miR-190a inhibition impairs insulin sensitivity, whereas its overexpression prevents the MGO-induced insulin resistance in MAECs. miR-190a levels are not affected by the inhibition of ERK1/2 phosphorylation. Conversely, ERK1/2 activation is sustained by miR-190a inhibitor and the MGO-induced ERK1/2 hyper-activation is reduced by miR-190a mimic transfection. Similarly, protein levels of the upstream KRAS are increased by both MGO and miR-190a inhibitor, and these levels are reduced by miR-190a mimic transfection. Interestingly, silencing of KRAS is able to rescue the MGO-impaired activation of IRS1/Akt/eNOS pathway in response to insulin.In conclusion, miR-190a down-regulation plays a role in MGO-induced endothelial insulin resistance by increasing KRAS. This study highlights miR-190a as new candidate for the identification of strategies aiming at ameliorating vascular function in diabetes.

Insulin Resistance May Contribute to Upregulation of Adhesion Molecules on Endothelial Cells in Psoriatic Plaques.

Psoriasis primarily affects the skin, but also has a systemic dimension and is associated with severe comorbidities. Since endothelial cells play an important role in psoriasis as well as in the development of cardiovascular comorbidities, we investigated whether a common mechanism, namely cytokine-induced insulin resistance, underlies both pathologies. Activation of the insulin pathway was studied in psoriatic skin and dermal endothelial cells. Expression of adhesion molecules was assessed by flow cytometry, as well as their biological function in flow chamber experiments. The phosphorylation status of Akt, a central kinase in the insulin pathway, suggests that endothelial cells within psoriatic plaques are rendered insulin resistant by pro-inflammatory cytokines. Insulin counteracts the expression of adhesion molecules, but has limited effects on interactions between T cells and endothelial cells. Pro-inflammatory cytokines induce insulin resistance in endothelial cells, which may contribute to the development of the inflammatory infiltrate in psoriasis.

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

Effect of metformin on insulin-resistant endothelial cell function.

The aim of the present study was to investigate the effect of metformin on the function of insulin-resistant (IR) endothelial cells. A model of IR endothelial cells was established by incubating cells with 30 mM glucose, 1 μM dexamethasone and various concentrations of insulin. The nitric oxide (NO) content of the endothelial cells was determined by measuring the rate of nitroreductase production; the endothelin (ET) concentration was examined by enzyme-linked immunosorbent assay; and the expression levels of endothelial nitric oxide synthase (eNOS) were detected using western blotting. The optimal conditions for inducing insulin resistance in endothelial cells were a combination treatment of 10−4 mmol/l insulin, 30 mM glucose and 1 μM dexamethasone for 48 h. Notably, metformin administration significantly increased the NO content and reduced the ET-1 concentration in the IR cells compared with the non-treated control cells (P<0.05); furthermore, metformin significantly increased the intracellular eNOS protein expression in IR endothelial cells compared with the non-treated control cells (P<0.05), with an optimal metformin concentration of 10−3 mmol/l. Thus, the present study identified that metformin improves the function of IR endothelial cells, possibly through promoting eNOS protein expression and increasing the NO content.

The endothelial protective effects of pioglitazone on insulin resistance in endothelial cells.

Insulin resistance plays an important role in vascular endothelial damage and atherosclerosis. Pioglitazone is an insulin-sensitizing agent and can reduce insulin resistance. In this study, the cellular model of insulin resistance was used to investigate the mechanisms involved in the endothelial protective effects of pioglitazone in a vascular endothelial cell damage model. The results showed that pioglitazone could effectively increase the survival rate of human umbilical vein endothelial cells (ECV), reduce apoptosis, and relieve insulin resistance damage. To understand the endothelial protective effect mechanisms of pioglitazone, we showed that 50 ng/mL and 100 ng/mL of pioglitazone could upregulate the levels of inducible nitric oxide synthase (NOS) and pioglitazone could induce NO levels. These results suggested that pioglitazone could have endothelial protective effects in a vascular endothelial cell damage model of insulin resistance and used to prevent beforehand and treat a vascular endothelial cell damage of insulin resistance.

Serine Phosphorylation Sites on IRS2 Activated by Angiotensin II and Protein Kinase C To Induce Selective Insulin Resistance in Endothelial Cells

Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells.

203 MUTATING THE IGF-1 ON THE VASCULAR ENDOTHELIUM: DIVERGENT EFFECTS ON WHOLE BODY AND ENDOTHELIAL INSULIN RESISTANCE

IntroductionEndothelial dysfunction is associated with the onset of atherosclerosis and cardiovascular disease. Whole body and endothelial cell insulin resistance can result in reduced nitric oxide (NO) bioavailability; this is a...

Palmitate induces phenotype changes in monocytes via de novo ceramide synthesis

Ageing is associated with physiological changes such as altered adipose tissue distribution and redox status, elevated metabolic disorder risk and increased free fatty acids. Saturated fatty acids can induce insulin resistance in endothelial cells and monocytes, in part explaining the increase cardiovascular disease risk with elevated fatty acids. The aim of this study is to investigate the effects of the two major physiological fatty acids palmitate and oleate (saturated and monounsaturated fatty acids respectively) on monocyte phenotype. THP-1 monocyte cell surface marker expression, mitochondrial reactive oxygen species, cell viability and caspase-3 were evaluated following 24h treatment with palmitate, oleate or bovine serum albumin. A concentration dependent increase in CD11b (p<0.01), CD36 (p<0.001) and mitochondrial reactive oxygen species (p<0.05) following palmitate but not oleate treatment was determined by flow cytometry. Decreased metabolic viability (p<0.01) was observed with palmitate (300μM), whilst no significant change in caspase-3 was observed. The superoxide dismutase mimetic MnTBAP (200μM) ameliorated the reduced metabolic viability and increased mitochondrial ROS due to palmitate, whilst increased CD11b and CD36 were unaffected. De novo ceramide synthesis inhibitor fumonisin B1 (50μM) prevented the palmitate dependent increase in CD11b (p<0.05) and CD36 (p<0.001). Ceramide converting inhibitors 1S,2R-D-erythro-2-N-myristoylamino)-1-phenyl-1-propanol (d-erythro MAPP 20 μM) and 1-phenyl-2-palmitoylamino-3-morpholino-1- propanol (PPMP 0.05 μM) were used to determine if ceramides or downstream sphingolipids are required for palmitate induced phenotype changes in monocytes. In the presence of MAPP and PPMP, palmitate induced increase in CD11b are sustained suggesting sphingolipids are key players in the observed phenotypic changes. The present study demonstrates in monocytes that palmitate but not oleate increases cell surface marker expression, accompanied by increased mitochondrial ROS and decreased metabolic viability. MnTBap could ameliorate changes in mitochondrial ROS and metabolic viability, but surface antigen expression decreased only by fumonisin B1 suggesting changes require de novo ceramide synthesi.

Positive and negative regulation of insulin action by genistein in the endothelium

Genistein is an isoflavone phytoestrogen with biological activities in management of metabolic disorders. This study aims to evaluate the regulation of insulin action by genistein in the endothelium. Genistein inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and attenuated downstream Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, leading to a decreased nitric oxide (NO) production in endothelial cells. These results demonstrated its negative regulation of insulin action in the endothelium. Palmitate (PA) stimulation evoked inflammation and induced insulin resistance in endothelial cells. Genistein inhibited IKKβ and nuclear factor-кB (NF-кB) activation with down-regulation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and expression. Genistein inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-mediated tyrosine phosphorylation. Genistein restored insulin-mediated Akt and eNOS phosphorylation, and then led to an increased NO production from endothelial cells, well demonstrating its positive regulation of insulin action under insulin-resistant conditions. Meanwhile, genistein effectively inhibited inflammation-enhanced mitogenic actions of insulin by down-regulation of endothelin-1 and vascular cell adhesion protein-1 overexpression. PA stimulation impaired insulin-mediated vessel dilation in rat aorta, while genistein effectively restored the lost vasodilation in a concentration-dependent manner (0.1, 1 and 10 μM). These results suggested that genistein inhibited inflammation and ameliorated endothelial dysfunction implicated in insulin resistance. Better understanding of genistein action in regulation of insulin sensitivity in the endothelium could be beneficial for its possible applications in controlling endothelial dysfunction associated with diabetes and insulin resistance.

D Endothelium specific insulin resistance leads to accelerated atherosclerosis: a role for reactive oxygen species

<h3>Background</h3> Global insulin resistance and endothelial dysfunction have been identified as predisposing factors for atherosclerosis. However, it is unclear whether selective insulin resistance in endothelial cells alone, is sufficient to promote atherosclerosis. We addressed this question by crossing Endothelial Specific Mutant Insulin Receptor Over-expressing (ESMIRO) mice with ApoE^−/− mice. ESMIRO mice over-express a human insulin receptor with an Ala-Thr1134 mutation in the tyrosine kinase domain (which disrupts insulin signalling) selectively in endothelial cells under the control of the tie-2 promoter/enhancer. <h3>Methods</h3> Male ApoE^−/−/ESMIRO mice were compared with sex-matched littermate ApoE^−/− mice (both on a C57Bl6 background) after feeding a Western-style diet for 12 weeks. <h3>Results</h3> ApoE^−/−/ESMIRO mice were morphologically indistinguishable from ApoE^−/− control littermates and showed normal development with no differences between groups in body mass. Heart rate, systolic blood pressure, glucose tolerance, insulin sensitivity and fasting glucose levels were similar in ApoE^−/−/ESMIRO and ApoE^−/− mice. ApoE^−/−/ESMIRO cultured endothelial cells demonstrated insulin resistance through significantly reduced insulin mediated eNOS activity (p=0.003). Aortic lipid deposition along the whole aorta, assessed by en-face oil red O staining, was similar in ApoE^−/−ESMIRO and ApoE^−/− mice (6.4%±0.5% vs 5.8%±0.5%; p=0.39). Analysis of lipid deposition along the lesser curvature of the aortic arch revealed a significant increase in ApoE^−/−ESMIRO when compared to controls (9.4±0.89 vs 12.43±1.19% p=0.035). Atherosclerotic lesion area in cross sections of aortic sinus was also significantly increased in ApoE^−/−/ESMIRO mice compared to ApoE^−/− controls (24.8%±2.4% vs 16.6%±2.4%; p=0.02). Vascular function assessed through relaxation responses of aortic rings in response to the endothelial specific vasodilator acetylcholine revealed that aortic rings from ApoE^−/−/ESMIRO mice had blunted relaxation responses to acetylcholine (Emax ApoE^−/− 102.88±6, Emax ApoE^−/−/ESMIRO 65±41%, p=0.02), which was restored by the superoxide dismutase mimetic and antioxidant MnTMPyP (Emax ApoE^−/−/ESMIRO without MnTMPyP 65±41%, with MnTMPyP 112±15% p=0.048). Endothelial cells from ApoE^−/−/ESMIRO mice had significantly increased basal generation of superoxide (1.87-fold increase compared to ApoE^−/− p&lt;0.05) which was blunted by the selective NADPH oxidase inhibitor gp91ds-tat (11% reduction ±0.02, p=0.03) and the non-selective NO synthase inhibitor L-NMMA (6% reduction ±0.01, p=0.03). <h3>Conclusions</h3> Endothelial specific insulin resistance is sufficient to promote atherosclerosis and increase lesion area in ApoE null mice potentially via the increased ROS displayed in this model. This suggests that enhancing endothelial insulin sensitivity may be an appropriate target to prevent atherosclerosis in insulin-resistant conditions.

139 Endothelial specific insulin resistance promotes the development of atherosclerosis

<h3>Background</h3> Global insulin resistance and endothelial dysfunction have been identified as predisposing factors for atherosclerosis. However, it is unclear whether selective insulin resistance in endothelial cells alone, is sufficient to promote atherosclerosis. Here we addressed this question by crossing Endothelial Specific Mutant Insulin Receptor Over-expressing (ESMIRO) mice with ApoE null mice. ESMIRO mice over-express a human insulin receptor with Ala-Thr1134 mutation in the tyrosine kinase domain (which disrupts insulin signalling) selectively in endothelial cells under the control of the tie-2 promoter/enhancer. <h3>Methods</h3> Male ApoE^−/−ESMIRO mice were compared with sex-matched littermate ApoE^−/− mice (both on a C57Bl6 background) after feeding a Western-style diet for 12 weeks. <h3>Results</h3> ApoE^−/−ESMIRO mice were morphologically indistinguishable from ApoE^−/− control littermates, with normal development and no difference between groups in body mass. Heart rate, systolic blood pressure, glucose tolerance, insulin sensitivity and fasting glucose levels were similar in ApoE-/-ESMIRO and ApoE^−/− mice. Aortic lipid deposition, assessed by en-face oil red O staining, was similar in ApoE^−/−ESMIRO and ApoE^−/− mice (6.4%±0.5% vs 5.8%±0.5%; p=0.39). However, atherosclerotic lesion area in cross sections of aortic sinus was significantly increased in ApoE^−/−ESMIRO mice compared to ApoE^−/− controls (24.8%±2.4% vs 16.6%±2.4%; p=0.02). Absolute plaque size was also significantly increased in ApoE^−/−ESMIRO mice compared to ApoE controls (226 448.9±16 154 μm² vs 149 424.41±24 221 μm²; p=0.01). <h3>Conclusions</h3> Endothelial specific insulin resistance is sufficient to promote atherosclerosis and increase lesion area in ApoE null mice. This suggests that enhancing endothelial insulin sensitivity may be an appropriate target to prevent atherosclerosis in insulin-resistant conditions.

Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A(2) mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser(1177) and Akt at Ser(473). These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser(380)/Thr(382/383) PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G(i) family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser(380)/Thr(382/383) phosphorylation, and dephosphorylation of both Akt (at Ser(473)) and eNOS (at Ser(1177)). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.