Vascular Endothelial Insulin Research Articles

Nebivolol Alleviates Vascular Endothelial Insulin Resistance by Inhibiting Endoplasmic Reticulum Stress.

Vascular endothelial insulin resistance (IR) is an important risk factor in the development of vascular complications in diabetes. Prolonged endoplasmic reticulum stress (ERS) contributes to the development and progression of endothelial IR. The current study assessed the effects and mechanism of nebivolol on vascular IR in Goto-Kakizaki (GK) rats and endothelial IR induced by high glucose (33.3 mmol/L) associated with high insulin (10-7 mol/L) in human aortic endothelial cells (HAECs). Rats were divided into Wistar, Wistar + Neb (Wistar rats treated with nebivolol, 10 mg/kg, ig), GK, and GK + Neb (GK rats treated with nebivolol, 10 mg/kg, ig). GK rats showed hyperglycemia, dyslipidemia, impaired glucose homeostasis, metabolic IR, reduced relaxation to insulin, and lower serum nitric oxide (NO) level. Treatment with nebivolol for 4 months ameliorated insulin's vasorelaxation and NO production, and relieved dyslipidemia in GK rats. Additionally, nebivolol increased glucose uptake and NO level in the endothelial IR group in vitro. Nebivolol increased aortic expressions of IRS-1/PI3K/Akt/eNOS relative proteins and GLUT4 and reduced expressions of ERS markers (ATF6, GRP78, and CHOP, p-JNK/JNK). Furthermore, both nebivolol and TUDCA (ERS inhibitor) alleviated the attenuated IRS-1PI3K/Akt/eNOS pathway and enhanced ERS in HAECs IR. Tunicamycin (ERS inducer) not only induced endothelial IR but also blocked nebivolol's alleviation on the IRS-1PI3K/Akt/eNOS pathway and ERS. Nebivolol ameliorated endothelial IR partially by inhibiting ERS and then regulating the IRS-1/PI3K/Akt/eNOS signal.

Interaction of FOXO1 and SUMOylated PPARγ1 induced by hyperlipidemia and hyperglycemia favors vascular endothelial insulin resistance and dysfunction

PPARγ1 and FOXO1 are the key transcription factors that regulate insulin sensitivity. We previously found that a small ubiquitin-related modifier of PPARγ1 at K77 (SUMOylation) favored endothelial insulin resistance (IR) induced by high fat/high glucose (HF/HG) administration. However, whether and how the crosstalk between SUMOylated PPARγ1 and FOXO1 would mediate the development of the endothelial IR and dysfunction remains unclear. Here, we emphasize how PPARγ1-K77 SUMOylation would interact with FOXO1 and participate in the development of the endothelial IR and dysfunction. Our results show that the combination of HF/HG and PPARγ1-K77 SUMOylation exhibits a synergistic deteriorative effect on the endothelial IR and dysfunction, presenting decreased NO levels and elevated ET-1 levels, weakened PI3K/Akt/eNOS signaling, and impaired endothelium-dependent vasodilation function. The further researches reveal that PPARγ1-K77 SUMOylation readily interacts with FOXO1, and FOXO1 occupies the PPAR response element (PPRE) which is supposed to be occupied by PPARγ, thus resulting in the decrease of PPARγ1 transcription activity and the mitigation of the PI3K/Akt signaling. Moreover, the mitigation of the PI3K/Akt signaling promotes in turn the accumulation of FOXO1 in the nucleus where FOXO1 interacts with the SUMOylated PPARγ1, thus exerting a positive feedback effect on IR pathogenesis. The findings uncover a novel association between PPARγ1-K77 SUMOylation and FOXO1, which contributes to our understanding of the pathogenesis of endothelial IR and dysfunction and provides novel pharmacological targets for diabetic angiopathy.

Interaction of FOXO1 and  SUMOylated PPARγ1 Induced by Hyperlipidemia and Hyperglycemia Favors Vascular Endothelial Insulin Resistance and Dysfunction

Interaction of FOXO1 and  SUMOylated PPARγ1 Induced by Hyperlipidemia and Hyperglycemia Favors Vascular Endothelial Insulin Resistance and Dysfunction

Macrophage Depletion Improves Endothelial Insulin Resistance and Protects against Cardiovascular Injury in Salt-Sensitive Hypertension.

Vascular endothelial insulin signaling is critical for the maintenance of vascular and metabolic homeostasis. We have previously shown that in hypertensive Dahl rats, impaired vascular insulin action is linked to angiotensin II activation of the NFκB inflammatory pathway. Macrophage polarization (M1) has implicated in hypertensive and metabolic diseases. Here, we investigated the effect of macrophage depletion using liposome-encapsulated clodronate (LEC) on endothelial insulin resistance and cardiovascular remodeling in Dahl salt-sensitive (DS) rats. High salt intake (HS) for 5 weeks increased systolic blood pressure (SBP: 192 ± 5 vs. 144 ± 4 mmHg in NS, p < 0.05), aortic and cardiac hypertrophy, cardiac fibrosis, and impaired acetylcholine- and insulin-induced vasorelaxation, accompanied by impaired insulin activation of endothelial nitric oxide synthases (eNOS)/NO signaling. HS rats had a significant increase in CD68 (a monocyte/macrophage marker) expression in the aorta and the heart. LEC reduced SBP (168 ± 5 mmHg, p < 0.05) and cardiovascular injury and improved acetylcholine- and insulin-mediated vasorelaxation and insulin signaling molecules with a reduction in the macrophage infiltration in the aorta and the heart. HS rats also manifested an increase in the aortic expressions of inflammatory cytokines, including the ratio of phosphorylated inhibitory kappa B (Iκb)/Iκb, tumor necrosis factor α, and phosphorylated c-Jun N-terminal kinase (JNK) and oxidative stress, which were reduced in HS/LEC rats. Our results suggest that in salt-sensitive hypertension, macrophage may importantly contribute to endothelial insulin resistance, vascular inflammation, and injury. These findings support the idea that macrophages may be a new target for immunotherapy of vasculopathy in hypertensive and metabolic disorders.

Quercetin improves endothelial insulin sensitivity in obese mice by inhibiting Drp1 phosphorylation at serine 616 and mitochondrial fragmentation.

Studies have shown that endothelial insulin resistance induced by oxidative stress contributes to vascular dysfunction in metabolic disorders. Quercetin, a natural antioxidant, has been recently shown to exert protective effects on endothelial function. However, the effects of quercetin on endothelial insulin resistance and its underlying mechanism are unclear. Here, we found that chronic oral treatment of obese mice with quercetin increased vascular endothelial insulin sensitivity, accompanied by alleviated mitochondrial fragmentation as revealed by confocal imaging. In addition, western blot analysis showed that quercetin treatment suppressed the levels of dynamin-related protein 1 (Drp1) and phosphorylation at serine 616 in endothelial cells of obese mice. Mechanistically, quercetin specifically suppressed Drp1 phosphorylation at serine 616, whereas it showed little effects on the Drp1 level and its phosphorylation at serine 637 in cultured endothelial cells under oxidative stress. Furthermore, our results also showed that quercetin suppressed Drp1 phosphorylation at serine 616 by inhibiting PKCδ as revealed by western blot analysis. Knockdown of PKCδ with siRNA alleviated the protective effects of quercetin on endothelial-mitochondrial dynamics and insulin sensitivity. These results suggest that chronic oral treatment with quercetin exerts endothelial protective effects through inhibition of PKCδ and the resultant mitochondrial fragmentation.

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.

Caveolin-1 phosphorylation regulates vascular endothelial insulin uptake and is impaired by insulin resistance in rats.

As insulin entry into muscle interstitium is rate-limiting for its overall peripheral action, defining the route and regulation of its entry is critical. Caveolin-1 is required for caveola formation in vascular endothelial cells (ECs) and for EC insulin uptake. Whether this requirement reflects simply the need for caveola availability or involves a more active role for caveolae/caveolin-1 is not known. Here, we examined the role of insulin-stimulated tyrosine 14 (Tyr(14))-caveolin-1 phosphorylation in mediating EC insulin uptake and the role of cellular Src-kinase (cSrc), TNF-α/IL-6 and high fat diet (HFD) in regulating this process. Freshly isolated ECs from normal or HFD-fed rats and/or cultured ECs were treated with FITC-labelled or regular insulin with or without a Src or phosphotidylinositol-3-kinase inhibitor, TNF-α or IL-6, or transfecting FLAG-tagged wild-type (WT) or mutant (Y14F) caveolin-1. Tyr(14)-caveolin-1/Tyr(416) cSrc phosphorylation and FITC-insulin uptake were quantified by immunostaining and/or western blots. Insulin stimulated Tyr(14)-caveolin-1 phosphorylation during EC insulin uptake. Inhibiting cSrc, but not phosphotidylinositol-3-kinase, reduced insulin-stimulated caveolin-1 phosphorylation. Furthermore, inhibiting cSrc reduced FITC-insulin uptake by ∼50%. Overexpression of caveolin-1Y14F inhibited, while overexpression of WT caveolin-1 increased, FITC-insulin uptake. Exposure of ECs to TNF-α or IL-6, or to 1-week HFD feeding eliminated insulin-stimulated caveolin-1 phosphorylation and inhibited FITC-insulin uptake to a similar extent. Insulin stimulation of its own uptake requires caveolin-1 phosphorylation and Src-kinase activity. HFD in vivo and proinflammatory cytokines in vitro both inhibit this process.

Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent nuclear factor-kappa B trans-repression pathway

Vascular endothelial insulin resistance (IR) is a critically initial factor in cardiocerebrovascular events resulted from diabetes and is becoming a worldwide public health issue. Thiazolidinediones (TZDs) are clinical insulin-sensitizers acting through a canonical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent insulin trans-activation pathway. However, it remains elusive whether there are other mechanisms. In current study, we investigated whether TZDs improve endothelial IR induced by high glucose concentration or hyperglycemia via a non-canonical PPARγ-dependent nuclear factor-kappa B (NF-κB) trans-repression pathway. Our results showed that pre-treatment with TZDs dramatically decrease the susceptibility of endothelial cell to IR, while post-treatment notably improve the endothelial IR both in vitro and in vivo. Moreover, TZDs substantially increase the levels of endothelial nitric oxide synthase (eNOS) and inhibitory κB alpha (IκBα), whereas decrease those of the phosphorylated inhibitory κB kinase alpha/beta (phosphor-IKKα/β) and the cytokines including tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cellular adhesion molecule-1 (sVCAM-1), suggesting that TZDs act indeed through a PPARγ-dependent NF-κB trans-repression pathway. These findings highlighted a non-canonical mechanism for TZDs to ameliorate endothelial IR which might provide a potential strategy to prevent and treat the diabetic vascular complications clinically.

ENDOTHELIAL-SPECIFIC OVEREXPRESSION OF MUTATED INSULIN-LIKE GROWTH FACTOR-1 RECEPTORS (IGF-1R) ENHANCES ENDOTHELIAL INSULIN SENSITIVITY AND VASCULAR HYDROGEN PEROXIDE GENERATION

Expression of Insulin-like growth factor-1 receptor (IGF-1R) and hybrid receptors formed between IGF-1R and insulin receptor (IR) in the vascular endothelium may be contributing factors to initiation of insulin resistance. The hypothesis that the IGF-1R acts as a negative regulator of insulin sensitivity and NO bioavailability in the endothelium is further investigated. Mice expressing non-functional, mutated human IGF-1R (K1003R) on the vascular endothelium (mIGFREO) under the control of Tie2 promoter-enhancer were generated. mIGFREO demonstrated no significant abnormalities in body and organ weight, as well as blood pressure compared to the wild type (WT) littermates. mIGFREO mice demonstrated preserved glucoregulation but showed a significant enhancement of whole body insulin sensitivity. Total hybrid expression was increased in the mIGFREO endothelial cells compared to WT controls, as well as the activated tyrosine phosphorylated insulin receptor (IRpY1328). Free fatty acid levels were lower in mIGFREO mice compared to WT littermates. Endothelial function assessed by vasorelaxation of aorta to acetylcholine remained unchanged between mIGFREO and WT littermates. The basal level of eNOS phosphorylation was enhanced in the mIGFREO endothelial cells compared to WT littermates. Nevertheless, mIGFREO aorta was significantly resistant to the vasodilatory effect of insulin. Interestingly, superoxide anion production was decreased among the mIGFREO mice compared to the WT littermate controls. Protein ex pression of NOX2, a source of superoxide anion generation was found to be significantly reduced, with a concomitant increase in NOX4 expression. These data suggest that mutation of endothelial IGF-1R demonstrates divergent effects on whole body and vascular endothelial insulin sensitivity.

Role of vascular endothelial insulin sensitisation in vascular repair in systemic insulin resistance

BackgroundInsulin resistance is an independent risk factor for cardiovascular disease. Murine models with global insulin receptor haploinsufficiency (IRKO) show endothelial dysfunction and impaired vascular repair, potentially related to reduced number and function of endothelial progenitor cells and bioavailability of nitric oxide. We hypothesise that endothelium-specific insulin sensitisation, in the context of global insulin resistance, could reverse these vascular abnormalities. Therefore, we have bred IRKO mice with HIRECO mice, which overexpress a human insulin receptor in endothelial cells under the transcriptional regulation of the Tie-2 promoter-enhancer. MethodsDouble-transgenic offspring from HIRECO mice crossed with IRKO mice (HIRECOxIRKO) were compared with IRKO mice after confirming genotype with PCR on DNA extracted from ear samples. Metabolic assessment was done with glucose-tolerance and insulin-tolerance tests, and ELISA was used to measure plasma insulin concentrations. Blood pressure (BP) was measured with tail cuff plethysmography. Femoral artery denuding injury was induced with an angioplasty guide-wire; 4 days later, vessels were explanted after perfusion with Evans blue dye to quantify endothelial denudation. Circulating progenitor cells (CPCs, positive for Sca-1/Flk-1) were counted in peripheral venous blood by fluorescence-activated cell sorting. Continuous data were expressed as means (SD) and analysed with paired and unpaired Student's t tests as appropriate (significance at p<0·05). A minimum of five of each mouse type was used in each comparison, unless otherwise stated. FindingsHIRECOxIRKO and IRKO mice had similar growth profiles (weight at 4 months 28·3 g [3·0] vs 27·4 [0·9], p=0·51) and systolic BP (98 mm Hg [12·4] vs 106 [10·4], p=0·28), with comparable insulin sensitivity on tolerance tests. Basal plasma insulin concentrations were also similar (0·70 ng/mL [0·50] vs 0·76 [0·47], p=0·78; n=7), though HIRECOxIRKO did not display the post prandial hyperinsulinaemia seen in IRKO mice previously and in this study. Vascular repair was significantly enhanced in HIRECOxIRKO after femoral artery wire injury (HIRECOxIRKO 56·9% regeneration [11·1] vs IRKO 46·0% [5·1], p=0·048). This repair was not associated with any improvement in numbers of CPCs, which were similar in the two groups of mice (HIRECOxIRKO 90 Sca-1/Flk-1 positive cells [27] vs IRKO 105 [42], p=0·99]. InterpretationIn HIRECOxIRKO mice, restoration of vascular repair occurred after endothelial wire injury, which has been previously demonstrated as impaired in IRKO mice. This restoration happened even with comparable glucoregulation and BP, suggesting that the effect is related specifically to endothelial insulin sensitisation despite insulin resistance in traditional target tissues. These data are early and exploratory, and sample size was small and from murine colonies; nonetheless, they seem to support the possibility of vascular endothelial insulin sensitisation as a potential therapeutic target in insulin resistant states. A mechanism remains to be established and we are planning assays of endothelial function, angiogenesis, and downstream insulin molecular signalling. FundingBritish Heart Foundation.

163 VASCULAR ENDOTHELIAL INSULIN SENSITISATION REDUCES BLOOD PRESSURE AND PROMOTES ENDOTHELIAL REPAIR IN THE CONTEXT OF GLOBAL INSULIN RESISTANCE

Introduction Insulin resistant syndromes are associated with an increased risk of sustaining major cardiovascular events; diminished endogenous vascular endothelial repair may contribute to this phenomenon. It remains unclear whether insulin sensitisation is a valid target to reduce cardiovascular events. Methods Global insulin receptor haploinsufficient mice (IRKO; n=8) were crossed with mice overexpressing the human insulin receptor under the transcriptional regulation of the Tie2 promoter-enhancer (IRKOxHIRECO; n=8) in order to augment vascular endothelial insulin sensitivity. Metabolic assessment was performed by assay of fasting glucose and insulin, followed by glucose- and insulin-tolerance tests. Blood pressure was assessed with tail cuff plethysmography. Femoral artery denuding injury was induced with an angioplasty guidewire and sham surgery performed contralaterally; 4 days later vessels were explanted after Evans blue dye perfusion in order to assess persistent endothelial denudation. Results IRKOxHIRECO exhibited weight gain (28.3 [1.2] versus 27.4 [0.4] g at 4 months of age; p=NS), basal plasma glucose (6.9 [0.5] versus 7.0 [0.4] mmol/L; p=NS) and basal serum insulin (0.74 [0.13] versus 0.66 [0.16] ng/ml; p=NS) concentrations comparable to IRKO; glucose tolerance testing and insulin tolerance testing also suggested comparable metabolic insulin sensitivity. However, IRKOxHIRECO mice demonstrated reduced systolic blood pressure when compared with IRKO (mean 101.0 [SEM 0.6] versus 110.1 [0.6] mmHg; p Conclusions Restoration of vascular endothelial insulin signalling in the context of global insulin resistance reduces blood pressure and augments vascular endothelial regeneration after denuding injury. These data support the pursuit of vascular insulin sensitising strategies as a means of promoting vascular repair.

Vascular endothelial insulin/IGF-1 signaling controls skin wound vascularization

Type 2 diabetes mellitus affects 6% of western populations and represents a major risk factor for the development of skin complications, of which impaired wound healing, manifested in e.g. “diabetic foot ulcer”, is most prominent. Impaired angiogenesis is considered a major contributing factor to these non-healing wounds. At present it is still unclear whether diabetes-associated wound healing and skin vascular dysfunction are direct consequences of impaired insulin/IGF-1 signaling, or secondary due to e.g. hyperglycemia. To directly test the role of vascular endothelial insulin signaling in the development of diabetes-associated skin complications and vascular function, we inactivated the insulin receptor and its highly related receptor, the IGF-1 receptor, specifically in the endothelial compartment of postnatal mice, using the inducible Tie-2CreERT (DKOIVE) deleter. Impaired endothelial insulin/IGF-1 signaling did not have a significant impact on endothelial homeostasis in the skin, as judged by number of vessels, vessel basement membrane staining intensity and barrier function. In contrast, challenging the skin through wounding strongly reduced neo-angiogenesis in DKOIVE mice, accompanied by reduced granulation tissue formation reduced. These results show that endothelial insulin/IGF signaling is essential for neo-angiogenesis upon wounding, and imply that reduced endothelial insulin/IGF signaling directly contributes to diabetes-associated impaired healing.

Caveolin-1 is required for vascular endothelial insulin uptake

As insulin's movement from plasma to muscle interstitium is rate limiting for its metabolic action, defining the regulation of this movement is critical. Here, we address whether caveolin-1 is required for the first step of insulin's transendothelial transport, its uptake by vascular endothelial cells (ECs), and whether IL-6 and TNFα affect insulin uptake or caveolin-1 expression. Uptake of FITC-labeled insulin was measured using confocal microscopy in control bovine aortic ECs (bAECs), in bAECs in which caveolin-1 was either knocked down or overexpressed, in murine ECs from caveolin-1(-/-) mice and in bAECs exposed to inflammatory cytokines. Knockdown of caveolin-1 expression in bAECs using specific caveolin-1 siRNA reduced caveolin-1 mRNA and protein expression by ∼ 70%, and reduced FITC-insulin uptake by 67% (P < 0.05 for each). Over-expression of caveolin-1 increased insulin uptake (P < 0.05). Caveolin-1-null mouse aortic ECs did not take up insulin and re-expression of caveolin-1 by transfecting these cells with FLAG-tagged caveolin-1 DNA rescued FITC-insulin uptake. Knockdown of caveolin-1 significantly reduced both insulin receptor protein level and insulin-stimulated Akt1 phosphorylation. Knockdown of caveolin-1 also inhibited insulin-induced caveolin-1 and IGF-1 receptor translocation to the plasma membrane. Compared with controls, IL-6 or TNFα (20 ng/ml for 24 h) inhibited FITC-insulin uptake as well as the expression of caveolin-1 mRNA and protein (P < 0.05 for each). IL-6 or TNFα also significantly reduced plasma membrane-associated caveolin-1. Thus, we conclude that insulin uptake by ECs requires expression of caveolin-1 supporting a role for caveolae mediating insulin uptake. Proinflammatory cytokines may inhibit insulin uptake, at least in part, by inhibiting caveolin-1 expression.