Jessica Smith

The Insulin-Like Growth Factor-1 Receptor Is a Negative Regulator of Nitric Oxide Bioavailability and Insulin Sensitivity in the Endothelium

OBJECTIVE In mice, haploinsufficiency of the IGF-1 receptor (IGF-1R+/−), at a whole-body level, increases resistance to inflammation and oxidative stress, but the underlying mechanisms are unclear. We hypothesized that by forming insulin-resistant heterodimers composed of one IGF-1Rαβ and one insulin receptor (IR), IRαβ complex in endothelial cells (ECs), IGF-1R reduces free IR, which reduces EC insulin sensitivity and generation of the antioxidant/anti-inflammatory signaling radical nitric oxide (NO). RESEARCH DESIGN AND METHODS Using a number of complementary gene-modified mice with reduced IGF-1R at a whole-body level and specifically in EC, and complementary studies in EC in vitro, we examined the effect of changing IGF-1R/IR stoichiometry on EC insulin sensitivity and NO bioavailability. RESULTS IGF-1R+/− mice had enhanced insulin-mediated glucose lowering. Aortas from these mice were hypocontractile to phenylephrine (PE) and had increased basal NO generation and augmented insulin-mediated NO release from EC. To dissect EC from whole-body effects we generated mice with EC-specific knockdown of IGF-1R. Aortas from these mice were also hypocontractile to PE and had increased basal NO generation. Whole-body and EC deletion of IGF-1R reduced hybrid receptor formation. By reducing IGF-1R in IR-haploinsufficient mice we reduced hybrid formation, restored insulin-mediated vasorelaxation in aorta, and insulin stimulated NO release in EC. Complementary studies in human umbilical vein EC in which IGF-1R was reduced using siRNA confirmed that reducing IGF-1R has favorable effects on NO bioavailability and EC insulin sensitivity. CONCLUSIONS These data demonstrate that IGF-1R is a critical negative regulator of insulin sensitivity and NO bioavailability in the endothelium.

Insulin Resistance Impairs Circulating Angiogenic Progenitor Cell Function and Delays Endothelial Regeneration

OBJECTIVE Circulating angiogenic progenitor cells (APCs) participate in endothelial repair after arterial injury. Type 2 diabetes is associated with fewer circulating APCs, APC dysfunction, and impaired endothelial repair. We set out to determine whether insulin resistance adversely affects APCs and endothelial regeneration. RESEARCH DESIGN AND METHODS We quantified APCs and assessed APC mobilization and function in mice hemizygous for knockout of the insulin receptor (IRKO) and wild-type (WT) littermate controls. Endothelial regeneration after femoral artery wire injury was also quantified after APC transfusion. RESULTS IRKO mice, although glucose tolerant, had fewer circulating Sca-1+/Flk-1+ APCs than WT mice. Culture of mononuclear cells demonstrated that IRKO mice had fewer APCs in peripheral blood, but not in bone marrow or spleen, suggestive of a mobilization defect. Defective vascular endothelial growth factor–stimulated APC mobilization was confirmed in IRKO mice, consistent with reduced endothelial nitric oxide synthase (eNOS) expression in bone marrow and impaired vascular eNOS activity. Paracrine angiogenic activity of APCs from IRKO mice was impaired compared with those from WT animals. Endothelial regeneration of the femoral artery after denuding wire injury was delayed in IRKO mice compared with WT. Transfusion of mononuclear cells from WT mice normalized the impaired endothelial regeneration in IRKO mice. Transfusion of c-kit+ bone marrow cells from WT mice also restored endothelial regeneration in IRKO mice. However, transfusion of c-kit+ cells from IRKO mice was less effective at improving endothelial repair. CONCLUSIONS Insulin resistance impairs APC function and delays endothelial regeneration after arterial injury. These findings support the hypothesis that insulin resistance per se is sufficient to jeopardize endogenous vascular repair. Defective endothelial repair may be normalized by transfusion of APCs from insulin-sensitive animals but not from insulin-resistant animals.

Increasing Circulating IGFBP1 Levels Improves Insulin Sensitivity, Promotes Nitric Oxide Production, Lowers Blood Pressure, and Protects Against Atherosclerosis

Low concentrations of insulin-like growth factor (IGF) binding protein-1 (IGFBP1) are associated with insulin resistance, diabetes, and cardiovascular disease. We investigated whether increasing IGFBP1 levels can prevent the development of these disorders. Metabolic and vascular phenotype were examined in response to human IGFBP1 overexpression in mice with diet-induced obesity, mice heterozygous for deletion of insulin receptors (IR+/−), and ApoE−/− mice. Direct effects of human (h)IGFBP1 on nitric oxide (NO) generation and cellular signaling were studied in isolated vessels and in human endothelial cells. IGFBP1 circulating levels were markedly suppressed in dietary-induced obese mice. Overexpression of hIGFBP1 in obese mice reduced blood pressure, improved insulin sensitivity, and increased insulin-stimulated NO generation. In nonobese IR+/− mice, overexpression of hIGFBP1 reduced blood pressure and improved insulin-stimulated NO generation. hIGFBP1 induced vasodilatation independently of IGF and increased endothelial NO synthase (eNOS) activity in arterial segments ex vivo, while in endothelial cells, hIGFBP1 increased eNOS Ser1177 phosphorylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE−/− mice, overexpression of hIGFBP1 reduced atherosclerosis. These favorable effects of hIGFBP1 on insulin sensitivity, blood pressure, NO production, and atherosclerosis suggest that increasing IGFBP1 concentration may be a novel approach to prevent cardiovascular disease in the setting of insulin resistance and diabetes.

Endothelium-specific insulin resistance leads to accelerated atherosclerosis in areas with disturbed flow patterns: A role for reactive oxygen species

OBJECTIVEnSystemic insulin resistance is associated with a portfolio of risk factors for atherosclerosis development. We sought to determine whether insulin resistance specifically at the level of the endothelium promotes atherosclerosis and to examine the potential involvement of reactive oxygen species.nnnMETHODSnWe cross-bred mice expressing a dominant negative mutant human insulin receptor specifically in the endothelium (ESMIRO) with ApoE(-/-) mice to examine the effect of endothelium-specific insulin resistance on atherosclerosis.nnnRESULTSnApoE(-/-)/ESMIRO mice had similar blood pressure, plasma lipids and whole-body glucose tolerance, but blunted endothelial insulin signalling, in comparison to ApoE(-/-) mice. Atherosclerosis was significantly increased in ApoE(-/-)/ESMIRO mice at the aortic sinus (226 ± 16 versus 149 ± 24 × 10(3) μm(2), P = 0.01) and lesser curvature of the aortic arch (12.4 ± 1.2% versus 9.4 ± 0.9%, P = 0.035). Relaxation to acetylcholine was blunted in aorta from ApoE(-/-)/ESMIRO mice (Emax 65 ± 41% versus 103 ± 6%, P = 0.02) and was restored by the superoxide dismutase mimetic MnTMPyP (Emax 112 ± 15% versus 65 ± 41%, P = 0.048). Basal generation of superoxide was increased 1.55 fold (P = 0.01) in endothelial cells from ApoE(-/-)/ESMIRO mice and was inhibited by the NADPH oxidase inhibitor gp91ds-tat (-12 ± 0.04%, P = 0.04), the NO synthase inhibitor L-NMMA (-8 ± 0.02%, P = 0.001) and the mitochondrial specific inhibitor rotenone (-23 ± 0.04%, P = 0.006).nnnCONCLUSIONSnInsulin resistance specifically at the level of the endothelium leads to acceleration of atherosclerosis in areas with disturbed flow patterns such as the aortic sinus and the lesser curvature of the aorta. We have identified a potential role for increased generation of reactive oxygen species from multiple enzymatic sources in promoting atherosclerosis in this setting.

Haploinsufficiency of the Insulin-Like Growth Factor-1 Receptor Enhances Endothelial Repair and Favorably Modifies Angiogenic Progenitor Cell Phenotype

Objectives— Defective endothelial regeneration predisposes to adverse arterial remodeling and is thought to contribute to cardiovascular disease in type 2 diabetes mellitus. We recently demonstrated that the type 1 insulin-like growth factor receptor (IGF1R) is a negative regulator of insulin sensitivity and nitric oxide bioavailability. In this report, we examined partial deletion of the IGF1R as a potential strategy to enhance endothelial repair. Approach and Results— We assessed endothelial regeneration after wire injury in mice and abundance and function of angiogenic progenitor cells in mice with haploinsufficiency of the IGF1R (IGF1R+/−). Endothelial regeneration after arterial injury was accelerated in IGF1R+/− mice. Although the yield of angiogenic progenitor cells was lower in IGF1R+/− mice, these angiogenic progenitor cells displayed enhanced adhesion, increased secretion of insulin-like growth factor-1, and enhanced angiogenic capacity. To examine the relevance of IGF1R manipulation to cell-based therapy, we transfused IGF1R+/− bone marrow–derived CD117+ cells into wild-type mice. IGF1R+/− cells accelerated endothelial regeneration after arterial injury compared with wild-type cells and did not alter atherosclerotic lesion formation. Conclusions— Haploinsufficiency of the IGF1R is associated with accelerated endothelial regeneration in vivo and enhanced tube forming and adhesive potential of angiogenic progenitor cells in vitro. Partial deletion of IGF1R in transfused bone marrow–derived CD117+ cells enhanced their capacity to promote endothelial regeneration without altering atherosclerosis. Our data suggest that manipulation of the IGF1R could be exploited as novel therapeutic approach to enhance repair of the arterial wall after injury.

Endothelial Insulin Receptor Restoration Rescues Vascular Function in Male Insulin Receptor Haploinsufficient Mice

Abstract Reduced systemic insulin signaling promotes endothelial dysfunction and diminished endogenous vascular repair. We investigated whether restoration of endothelial insulin receptor expression could rescue this phenotype. Insulin receptor knockout (IRKO) mice were crossed with mice expressing a human insulin receptor endothelial cell–specific overexpression (hIRECO) to produce IRKO-hIRECO progeny. No metabolic differences were noted between IRKO and IRKO-hIRECO mice in glucose and insulin tolerance tests. In contrast with control IRKO littermates, IRKO-hIRECO mice exhibited normal blood pressure and aortic vasodilatation in response to acetylcholine, comparable to parameters noted in wild type littermates. These phenotypic changes were associated with increased basal- and insulin-stimulated nitric oxide production. IRKO-hIRECO mice also demonstrated normalized endothelial repair after denuding arterial injury, which was associated with rescued endothelial cell migration in vitro but not with changes in circulating progenitor populations or culture-derived myeloid angiogenic cells. These data show that restoration of endothelial insulin receptor expression alone is sufficient to prevent the vascular dysfunction caused by systemically reduced insulin signaling.

197 Alpha-5 Beta-1 Integrin: a Promising Therapeutic Target in the Field of Insulin Resistance and Cardiovascular Disease

Obesity is a key factor in the development of insulin resistance. Resistance to the vascular effects of insulin plays a central role in the initiation and progression of cardiovascular disease. Developing novel therapeutic strategies to prevent and treat insulin resistance is therefore important. IGFBP1 (Insulin like growth factor binding protein 1) is a 30kDa protein, derived mainly from the liver. We have previously shown that in vivo over expression of IGFBP1 improves insulin sensitivity, promotes nitric oxide production, lowers blood pressure and protects against atherosclerosis. IGFBP1 can impact on cellular functions via an RGD (α5β1 integrin binding) motif independent of IGF binding. However, whether the integrin binding motif of IGFBP1 could be exploited therapeutically remained unexplored. The cell line C2C12, an insulin responsive skeletal muscle cell line, was used to investigate the effects with acute treatment of GRGDTP synthetic hexapeptide (which binds α5β1 integrin) on the insulin signalling pathway, through immunoblotting of key insulin signalling proteins – IR (Insulin receptor), IRS1 (Insulin receptor substrate 1) and AKT (Protein kinase B). To investigate the possible role of integrin activation, the effects of acute treatment of GRGDTP on FAK (Focal adhesion kinase) phosphorylation were also investigated. C2C12 cells were also used in a glucose uptake assay, to determine the effects of GRGDTP on insulin stimulated glucose uptake. To examine whether the RGD motif of IGFBP1 could be exploited therapeutically synthetic hexapeptide GRGDTP was administered to C57BL/6 mice with diet-induced obesity. Treatment with GRGDTP prior to insulin stimulation of C2C12 cells enhances FAK, IRS1 and AKT phosphorylation (P ≤ 0.05). Treatment with GRGDTP prior to insulin stimulation of C2C12 cells increased insulin stimulated glucose uptake (P ≤ 0.01). Acute administration of GRGDTP (40 μg IP) significantly improved glucose tolerance (P ≤ 0.05) and insulin sensitivity in C57BL/6 mice with diet-induced obesity (P ≤ 0.05). For the first time, we have shown that the integrin binding domain of IGFBP1 may play an important role in insulin sensitivity and glucose regulation and represents a promising therapeutic agent in the field of insulin resistance and cardiovascular disease.

184 Temporospatial Effects of Endothelium Specific Inhibition of Shc Homology 2 -containing Inositol 5´ Phosphatase 2 on Nitric Oxide Bioavailability and Whole Body Insulin Sensitivity

Introduction Ageing is an important risk factor for diabetes and cardiovascular disease. Integrity of the endothelium plays a critical role in cardiovascular pathophysiology. Insulin signalling in endothelial cells modulates the generation of nitric oxide and reactive oxygen species. Although the vascular implications of endothelial insulin resistance are well understood, the effect of enhanced endothelial insulin signalling on whole body glucose regulation and vascular function remains poorly characterised. We therefore generated mice with downregulation of the negative regulator of insulin signalling; SHIP2 in endothelial cells, to investigate whether enhanced insulin signalling restricted to the endothelium modulates vascular function and whole body glucose regulation. Methods We deleted exons 18–19 of the ship 2 gene using Cre-Lox technology under the control of the Tie2 promoter to generate a catalytically inactivate protein. Male mice heterozygous for the inactive protein (ECSHIP2KD) were compared with sex-matched littermate controls. Results ECSHIPKD exhibited normal development. At 8 weeks of age ECSHIP2KD mice displayed increased glucose tolerance after glucose challenge (P = 0.03) and improved insulin sensitivity (P = 0.02) after insulin challenge compared to controls. Surprisingly however, by 40 weeks of age this phenotype was reversed; ECSHIP2KDmice revealed significant insulin resistance after insulin challenge (P = <0.05). Euglycemic hyperinsulinemic clamping confirmed whole body insulin resistance (decreased glucose infusion rate of 26% P < 0.05). In young mice ex vivo aortic vasomotor studies in both controls and ECSHIPKD revealed similar contractile responses to phenylephrine and displayed decreased contraction after insulin incubation. Both groups displayed increased contraction after NO synthase inhibitor LNMMA incubation. However, at 40 weeks old in ECSHIP2KD mice the vasodilatory aortic ring response to insulin was abolished (Emax controls 0.59 ± 0.04g vs 0.47 ± 0.03g P = 0.04, ECSHIP2KD 0.64 ± 0.04g vs 0.63 ± 0.06g P = 0.9) and ECSHIP2KD displayed no increase in contraction to LNMMA incubation (Emax controls 0.59 ± 0.04g vs 0.82 ± 0.08g P = 0.02, ECSHIP2KD0.64 ± 0.04g vs 0.69 ± 0.07g P = 00.5) indicating insulin resistance and lower basal nitric oxide (NO) production. Western blots experiments performed on cultured endothelial cells from 40 week old mice reveal significantly increased level of endothelial nitric oxide synthase (eNOS) but a lack of eNOS phosphorylation after stimulation with insulin. Resistance to activation by insulin was also confirmed through 14C L-citrulline conversion assay. Conclusion Downregulation of SHIP2 augments whole body glucose disposal in young mice but attenuates whole body glucose disposal in older mice, our data suggest this may be mediated by changes in nitric oxide bioavailability and identifies novel spatial and temporal specific affects of the lipid phosphatase SHIP2.