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Shc homology 2-containing inositol 5' phosphatase-2 (SHIP2) is a lipid phosphatase that inhibits insulin signaling downstream of phosphatidylinositol 3-kinase (PI3K); its role in vascular function is poorly understood. To examine its role in endothelial cell (EC) biology, we generated mice with catalytic inactivation of one SHIP2 allele selectively in ECs (ECSHIP2 Δ/+ ). Hyperinsulinemic-euglycemic clamping studies revealed that ECSHIP2 Δ/+ was resistant to insulin-stimulated glucose uptake in adipose tissue and skeletal muscle compared with littermate controls. ECs from ECSHIP2 Δ/+ mice had increased basal expression and activation of PI3K downstream targets, including Akt and endothelial nitric oxide synthase, although incremental activation by insulin and shear stress was impaired. Insulin-mediated vasodilation was blunted in ECSHIP2 Δ/+ mice, as was aortic nitric oxide bioavailability. Acetylcholine-induced vasodilation was also impaired in ECSHIP2 Δ/+ mice, which was exaggerated in the presence of a superoxide dismutase/catalase mimetic. Superoxide abundance was elevated in ECSHIP2 Δ/+ ECs and was suppressed by PI3K and NADPH oxidase 2 inhibitors. These findings were phenocopied in healthy human ECs after SHIP2 silencing. Our data suggest that endothelial SHIP2 is required to maintain normal systemic glucose homeostasis and prevent oxidative stress-induced endothelial dysfunction., (© 2017 by the American Diabetes Association.)

Low circulating levels of insulin-like growth factor binding protein 1 (IGFBP-1) are associated with insulin resistance and predict the development of type 2 diabetes. IGFBP-1 can affect cellular functions independently of IGF binding through an Arg-Gly-Asp (RGD) integrin-binding motif. Whether causal mechanisms underlie the favorable association of high IGFBP-1 levels with insulin sensitivity and whether these could be exploited therapeutically remain unexplored. We used recombinant IGFBP-1 and a synthetic RGD-containing hexapeptide in complementary in vitro signaling assays and in vivo metabolic profiling in obese mice to investigate the effects of IGFBP-1 and its RGD domain on insulin sensitivity, insulin secretion, and whole-body glucose regulation. The RGD integrin-binding domain of IGFBP-1, through integrin engagement, focal adhesion kinase, and integrin-linked kinase, enhanced insulin sensitivity and insulin secretion in C2C12 myotubes and INS-1 832/13 pancreatic β-cells. Both acute administration and chronic infusion of an RGD synthetic peptide to obese C57BL/6 mice improved glucose clearance and insulin sensitivity. These favorable effects on metabolic homeostasis suggest that the RGD integrin-binding domain of IGFBP-1 may be a promising candidate for therapeutic development in the field of insulin resistance., (© 2017 by the American Diabetes Association.)

Circadian rhythms are integral to the normal functioning of numerous physiological processes. Evidence from human and mouse studies suggests that loss of rhythm occurs in obesity and cardiovascular disease and may be a neglected contributor to pathophysiology. Obesity has been shown to impair the circadian clock mechanism in liver and adipose tissue but its effect on cardiovascular tissues is unknown. We investigated the effect of diet-induced obesity in C57BL6J mice upon rhythmic transcription of clock genes and diurnal variation in vascular and metabolic systems. In obesity, clock gene function and physiological rhythms were preserved in the vasculature but clock gene transcription in metabolic tissues and rhythms of glucose tolerance and insulin sensitivity were blunted. The most pronounced attenuation of clock rhythm occurred in adipose tissue, where there was also impairment of clock-controlled master metabolic genes and both AMPK mRNA and protein. Across tissues, clock gene disruption was associated with local inflammation but diverged from impairment of insulin signaling. We conclude that vascular tissues are less sensitive to pathological disruption of diurnal rhythms during obesity than metabolic tissues and suggest that cellular disruption of clock gene rhythmicity may occur by mechanisms shared with inflammation but distinct from those leading to insulin resistance.

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance-induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance-mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance-related vascular disease.

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 Ser(1177) 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.

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.

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.

Objective: Insulin resistance is an independent risk factor for the development of cardiovascular atherosclerosis. A key step in the development of atherosclerosis is endothelial dysfunction, manifest by a reduction in bioactivity of nitric oxide (NO). Insulin resistance is associated with endothelial dysfunction; however, the mechanistic relationship between these abnormalities and the role of impaired endothelial insulin signaling versus global insulin resistance remains unclear., Research Design and Methods: To examine the effects of insulin resistance specific to the endothelium, we generated a transgenic mouse with endothelium-targeted overexpression of a dominant-negative mutant human insulin receptor (ESMIRO). This receptor has a mutation (Ala-Thr(1134)) in its tyrosine kinase domain that disrupts insulin signaling. Humans with the Thr(1134) mutation are insulin resistant. We performed metabolic and vascular characterization of this model., Results: ESMIRO mice had preserved glucose homeostasis and were normotensive. They had significant endothelial dysfunction as evidenced by blunted aortic vasorelaxant responses to acetylcholine (ACh) and calcium ionophore. Furthermore, the vascular action of insulin was lost in ESMIRO mice, and insulin-induced endothelial NO synthase (eNOS) phosphorylation was blunted. Despite this phenotype, ESMIRO mice demonstrate similar levels of eNOS mRNA and protein expression to wild type. ACh-induced relaxation was normalized by the superoxide dismutase mimetic, Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride. Endothelial cells of ESMIRO mice showed increased superoxide generation and increased mRNA expression of the NADPH oxidase isoforms Nox2 and Nox4., Conclusions: Selective endothelial insulin resistance is sufficient to induce a reduction in NO bioavailability and endothelial dysfunction that is secondary to increased generation of reactive oxygen species. This arises independent of a significant metabolic phenotype.

Proliferation of adipocyte precursors and their differentiation into mature adipocytes contributes to the development of obesity in mammals. IGF-I is a potent mitogen and important stimulus for adipocyte differentiation. The biological actions of IGFs are closely regulated by a family of IGF-binding proteins (IGFBPs), which exert predominantly inhibitory effects. IGFBP-2 is the principal binding protein secreted by differentiating white preadipocytes, suggesting a potential role in the development of obesity. We have generated transgenic mice overexpressing human IGFBP-2 under the control of its native promoter, and we show that overexpression of IGFBP-2 is associated with reduced susceptibility to obesity and improved insulin sensitivity. Whereas wild-type littermates developed glucose intolerance and increased blood pressure with aging, mice overexpressing IGFBP-2 were protected. Furthermore, when fed a high-fat/high-energy diet, IGFBP-2-overexpressing mice were resistant to the development of obesity and insulin resistance. This lean phenotype was associated with decreased leptin levels, increased glucose sensitivity, and lower blood pressure compared with wild-type animals consuming similar amounts of high-fat diet. Our in vitro data suggest a direct effect of IGFBP-2 preventing adipogenesis as indicated by the ability of recombinant IGFBP-2 to impair 3T3-L1 differentiation. These findings suggest an important, novel role for IGFBP-2 in obesity prevention.

Previous studies have suggested an involvement of inducible nitric oxide synthase (iNOS) in obesity, but the relation, if any, between this and mechanisms underlying endothelial dysfunction in obesity is unknown. We studied mice fed an obesogenic high-fat or standard diet for up to 8 weeks. Obesity was associated with elevated blood pressure; resistance to the glucoregulatory actions of insulin; resistance to the vascular actions of insulin, assessed as the reduction in phenylephrine constrictor response of aortic rings after insulin preincubation (lean -21.7 +/- 11.5 vs. obese 18.2 +/- 15.5%; P < 0.05); and evidence of reactive oxygen species (ROS)-dependent vasodilatation in response to acetylcholine in aortic rings (change in maximal relaxation to acetylcholine after exposure to catalase: lean -2.1 +/- 6.0 vs. obese -15.0 +/- 3.8%; P = 0.04). Obese mice had increased expression of iNOS in aorta, with evidence of increased vascular NO production, assessed as the increase in maximal constriction to phenylephrine after iNOS inhibition with 1400W (lean -3.5 +/- 9.1 vs. obese 42.1 +/- 11.2%; P < 0.001). To further address the role of iNOS in obesity-induced vascular and metabolic dysfunction, we studied the effect of a high-fat diet in iNOS knockout mice (iNOS KO). Obese iNOS KO mice were protected against the development of resistance to insulin's glucoregulatory and vascular effects (insulin-dependent reduction in maximal phenylephrine response: obese wild-type 11.2 +/- 15.0 vs. obese iNOS KO -20.0 +/- 7.7%; P = 0.02). However, obese iNOS KO mice remained hypertensive (124.0 +/- 0.7 vs. 114.9 +/- 0.5 mmHg; P < 0.01) and had evidence of increased vascular ROS production. Although these data support iNOS as a target to protect against the adverse effects of obesity on glucoregulation and vascular insulin resistance, iNOS inhibition does not prevent the development of raised blood pressure or oxidative stress.

Type 2 diabetes is preceded by years of insulin resistance and is characterized by reduced bioavailability of the antiatherosclerotic signaling molecule nitric oxide (NO) and premature atherosclerosis. The relationship between resistance to the glucoregulatory actions of insulin and its effects on the vasculature (in particular NO-dependent responses) is poorly characterized. We studied this relationship in mice heterozygous for knockout of the insulin receptor (IRKO), which have a mild perturbation of insulin signaling. Male heterozygous IRKO mice aged 8-12 weeks were compared with age- and sex-matched littermates. IRKO mice had fasting blood glucose, insulin, free fatty acid, and triglyceride levels similar to those of wild-type mice. Intraperitoneal glucose and insulin tolerance tests were also similar in the two groups. Insulin levels in response to a glucose load were approximately twofold higher in IRKO compared with wild-type mice (1.08 +/- 0.11 vs. 0.62 +/- 0.13 ng/ml; P = 0.004). Despite this mild metabolic phenotype, IRKO mice had increased systolic blood pressure (124 +/- 4 vs. 110 +/- 3 mmHg; P = 0.01). Basal NO bioactivity, assessed from the increase in tension of phenylephrine preconstricted aortic rings in response to the NO synthase inhibitor N(G)-monomethyl-l-arginine, was reduced in IRKO (61 +/- 14 vs. 152 +/- 30%; P = 0.005). Insulin-mediated NO release in aorta, assessed as the reduction in phenylephrine constrictor response after insulin preincubation, was lost in IRKO mice (5 +/- 8% change vs. 66 +/- 9% reduction in wild-type; P = 0.03). Insulin-stimulated aortic endothelial NO synthase phosphorylation was also significantly blunted in IRKO mice (P < 0.05). These data demonstrate that insulin-stimulated NO responses in the vasculature are exquisitely sensitive to changes in insulin-signaling pathways in contrast to the glucoregulatory actions of insulin. These findings underscore the importance of early intervention in insulin-resistant states, where glucose homeostasis may be normal but substantial abnormalities of the vascular effects of insulin may already be present.

IGFs and their binding proteins (IGFBPs) play a significant role in metabolic regulation, and there is growing evidence that they also exert important vascular effects. IGFBP-1 contributes to glucose counterregulation, and observational studies demonstrate an inverse association between circulating IGFBP-1 levels and cardiovascular risk factors. Furthermore, IGFBP-1 levels are lower in subjects with overt macrovascular disease. We therefore hypothesized that IGFBP-1 exerts potentially beneficial effects, either directly or indirectly, on blood pressure regulation and vascular function. We tested this hypothesis using a unique transgenic mouse, which overexpresses human IGFBP-1, and explored the effect of this protein on metabolic, blood pressure, and vascular homeostasis. IGFBP-1-overexpressing mice exhibited postprandial hyperinsulinemia with preservation of glucocompetence and insulin sensitivity. Blood pressure was unchanged in the fasting state but was significantly lower in transgenic mice after a carbohydrate load. Aortic rings from IGFBP-1-overexpressing mice were hypocontractile in response to vasoconstrictors, and relaxation responses were unimpaired. Basal nitric oxide production was increased and endothelial nitric oxide synthase mRNA expression upregulated in aortae of these mice. Our data suggest that IGFBP-1 plays an important and potentially beneficial role in regulating metabolic and vascular homeostasis.