Effects of Quinidine or Rifampin Co-administration on the Single-Dose Pharmacokinetics and Safety of Rilzabrutinib (PRN1008) in Healthy Participants.

This open-label, phase 1 study was conducted with healthy adult participants to evaluate the potential drug-drug interaction between rilzabrutinib and quinidine (an inhibitor of P-glycoprotein [P-gp] and CYP2D6) or rifampin (an inducer of CYP3A and P-gp). Plasma concentrations of rilzabrutinib were measured after a single oral dose of rilzabrutinib 400 mg administered on day 1 and again, following a wash-out period, after co-administration of rilzabrutinib and quinidine or rifampin. Specifically, quinidine was given at a dose of 300 mg every 8 hours for 5 days from day 7 to day 11 (N = 16) while rifampin was given as 600 mg once daily for 11 days from day 7 to day 17 (N = 16) with rilzabrutinib given in the morning of day 10 (during quinidine dosing) or day 16 (during rifampin dosing). Quinidine had no significant effect on rilzabrutinib pharmacokinetics. Rifampin decreased rilzabrutinib exposure (the geometric mean of Cmax and AUC0-∞ decreased by 80.5% and 79.5%, respectively). Single oral doses of rilzabrutinib, with or without quinidine or rifampin, appeared to be well tolerated. These findings indicate that rilzabrutinib is a substrate for CYP3A but not a substrate for P-gp.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Homozygous receptors for insulin and not IGF-1 accelerate intimal hyperplasia in insulin resistance and diabetes.

Insulin and IGF-1 actions in vascular smooth muscle cells (VSMC) are associated with accelerated arterial intima hyperplasia and restenosis after angioplasty, especially in diabetes. To distinguish their relative roles, we delete insulin receptor (SMIRKO) or IGF-1 receptor (SMIGF1RKO) in VSMC and in mice. Here we report that intima hyperplasia is attenuated in SMIRKO mice, but not in SMIGF1RKO mice. In VSMC, deleting IGF1R increases homodimers of IR, enhances insulin binding, stimulates p-Akt and proliferation, but deleting IR decreases responses to insulin and IGF-1. Studies using chimeras of IR(extracellular domain)/IGF1R(intracellular-domain) or IGF1R(extracellular domain)/IR(intracellular-domain) demonstrate homodimer IRα enhances insulin binding and signaling which is inhibited by IGF1Rα. RNA-seq identifies hyaluronan synthase2 as a target of homo-IR, with its expression increases by IR activation in SMIGF1RKO mice and decreases in SMIRKO mice. Enhanced intima hyperplasia in diabetes is mainly due to insulin signaling via homo-IR, associated with increased Has2 expression.

Insulin transport across the blood-brain barrier can occur independently of the insulin receptor.

KEY POINTS: Insulin enters the brain from the blood via a saturable transport system. It is unclear how insulin is transported across the blood-brain barrier (BBB). Using two models of the signalling-related insulin receptor loss or inhibition, we show insulin transport can occur in vivo without the signalling-related insulin receptor. Insulin in the brain has multiple roles including acting as a metabolic regulator and improving memory. Understanding how insulin is transported across the BBB will aid in developing therapeutics to further increase CNS concentrations. ABSTRACT: A saturable system transports insulin from blood across the blood-brain barrier (BBB) and into the central nervous system. Whether or not the classic or signalling-related insulin receptor plays a role in mediating this transport in vivo is controversial. Here, we employed kinetics methods that distinguish between transport across the brain endothelial cell and reversible luminal surface receptor binding. Using a previously established line of mice with endothelial-specific loss of the signalling-related insulin receptor (EndoIRKO) or inhibiting the insulin receptor with the selective antagonist S961, we show insulin transport across the BBB is maintained. Rates of insulin transport were similar in all groups and transport was still saturable. Unlike transport, binding of insulin to the brain endothelial cell was decreased with the loss or inhibition of the signalling-related insulin receptor. These findings demonstrate that the signalling-related insulin receptor is not required for insulin transport across the BBB.

Exogenous Insulin Infusion Can Decrease Atherosclerosis in Diabetic Rodents by Improving Lipids, Inflammation, and Endothelial Function.

OBJECTIVE: The objective of this study is to evaluate whether exogenously induced hyperinsulinemia may increase the development of atherosclerosis. APPROACH AND RESULTS: Hyperinsulinemia, induced by exogenous insulin implantation in high-fat fed (60% fat HFD) apolipoprotein E-deficient mice (ApoE-/-) mice, exhibited insulin resistance, hyperglycemia, and hyperinsulinemia. Atherosclerosis was measured by the accumulation of fat, macrophage, and extracellular matrix in the aorta. After 8 weeks on HFD, ApoE-/- mice were subcutaneously implanted with control (sham) or insulin pellet, and phlorizin, a sodium glucose cotransporters inhibitor (1/2)inhibitor, for additional 8 weeks. Intraperitoneal glucose tolerance test showed that plasma glucose levels were lower and insulin and IGF-1 (insulin-like growth factor-1) levels were 5.3- and 3.3-fold higher, respectively, in insulin-implanted compared with sham-treated ApoE-/- mice. Plasma triglyceride, cholesterol, and lipoprotein levels were decreased in mice with insulin implant, in parallel with increased lipoprotein lipase activities. Atherosclerotic plaque by en face and complexity staining showed significant reductions of fat deposits and expressions of vascular adhesion molecule-1, tumor necrosis factor-α, interleukin 6, and macrophages in arterial wall while exhibiting increased activation of pAKT and endothelial nitric oxide synthase (P<0.05) comparing insulin-implanted versus sham HFD ApoE-/- mice. No differences were observed in atherosclerotic plaques between phlorizin-treated and sham HFD ApoE-/- mice, except phlorizin significantly lowered plasma glucose and glycated hemoglobin levels while increased glucosuria. Endothelial function was improved only by insulin treatment through endothelial nitric oxide synthase/nitric oxide activations and reduced proinflammatory (M1) and increased anti-inflammatory (M2) macrophages, which were inhibited by endothelial nitric oxide synthase inhibitor. CONCLUSIONS: Exogenous insulin decreased atherosclerosis by lowering inflammatory cytokines, macrophages, and plasma lipids in HFD-induced hyperlipidemia, insulin resistant and mildly diabetic ApoE-/- mice.

Endothelial insulin receptors differentially control insulin signaling kinetics in peripheral tissues and brain of mice.

Insulin receptors (IRs) on endothelial cells may have a role in the regulation of transport of circulating insulin to its target tissues; however, how this impacts on insulin action in vivo is unclear. Using mice with endothelial-specific inactivation of the IR gene (EndoIRKO), we find that in response to systemic insulin stimulation, loss of endothelial IRs caused delayed onset of insulin signaling in skeletal muscle, brown fat, hypothalamus, hippocampus, and prefrontal cortex but not in liver or olfactory bulb. At the level of the brain, the delay of insulin signaling was associated with decreased levels of hypothalamic proopiomelanocortin, leading to increased food intake and obesity accompanied with hyperinsulinemia and hyperleptinemia. The loss of endothelial IRs also resulted in a delay in the acute hypoglycemic effect of systemic insulin administration and impaired glucose tolerance. In high-fat diet-treated mice, knockout of the endothelial IRs accelerated development of systemic insulin resistance but not food intake and obesity. Thus, IRs on endothelial cells have an important role in transendothelial insulin delivery in vivo which differentially regulates the kinetics of insulin signaling and insulin action in peripheral target tissues and different brain regions. Loss of this function predisposes animals to systemic insulin resistance, overeating, and obesity.

Regulation of Macrophage Apoptosis and Atherosclerosis by Lipid-Induced PKCδ Isoform Activation.

RATIONALE: Activation of monocytes/macrophages by hyperlipidemia associated with diabetes mellitus and obesity contributes to the development of atherosclerosis. PKCδ (protein kinase C δ) expression and activity in monocytes were increased by hyperlipidemia and diabetes mellitus with unknown consequences to atherosclerosis. OBJECTIVE: To investigate the effect of PKCδ activation in macrophages on the severity of atherosclerosis. METHODS AND RESULTS: PKCδ expression and activity were increased in Zucker diabetic rats. Mice with selective deletion of PKCδ in macrophages were generated by breeding PKCδ flox/flox mice with LyzM-Cre and ApoE-/- mice (MPKCδKO/ApoE-/- mice) and studied in atherogenic (AD) and high-fat diet (HFD). Mice fed AD and HFD exhibited hyperlipidemia, but only HFD-fed mice had insulin resistance and mild diabetes mellitus. Surprisingly, MPKCδKO/ApoE-/- mice exhibited accelerated aortic atherosclerotic lesions by 2-fold versus ApoE-/- mice on AD or HFD. Splenomegaly was observed in MPKCδKO/ApoE-/- mice on AD and HFD but not on regular chow. Both the AD or HFD increased macrophage number in aortic plaques and spleen by 1.7- and 2-fold, respectively, in MPKCδKO/ApoE-/- versus ApoE-/- mice because of decreased apoptosis (62%) and increased proliferation (1.9-fold), and not because of uptake, with parallel increased expressions of inflammatory cytokines. Mechanisms for the increased macrophages in MPKCδKO/ApoE-/- were associated with elevated phosphorylation levels of prosurvival cell-signaling proteins, Akt and FoxO3a, with reduction of proapoptotic protein Bim associated with PKCδ induced inhibition of P85/PI3K. CONCLUSIONS: Accelerated development of atherosclerosis induced by insulin resistance and hyperlipidemia may be partially limited by PKCδ isoform activation in the monocytes, which decreased its number and inflammatory responses in the arterial wall.

SHP-1 activation inhibits vascular smooth muscle cell proliferation and intimal hyperplasia in a rodent model of insulin resistance and diabetes.

AIMS/HYPOTHESIS: Accelerated migration and proliferation of vascular smooth muscle cells (VSMCs) enhances arterial restenosis after angioplasty in insulin resistance and diabetes. Elevation of Src homology 2-containing protein tyrosine phosphatase 1 (SHP-1) induces apoptosis in the microvasculature. However, the role of SHP-1 in intimal hyperplasia and restenosis has not been clarified in insulin resistance and diabetes. METHODS: We used a femoral artery wire injury mouse model, rodent models with insulin resistance and diabetes, and patients with type 2 diabetes. Further, we modulated SHP-1 expression using a transgenic mouse that overexpresses SHP-1 in VSMCs (Shp-1-Tg). SHP-1 agonists were also employed to study the molecular mechanisms underlying the regulation of SHP-1 by oxidised lipids. RESULTS: Mice fed a high-fat diet (HFD) exhibited increased femoral artery intimal hyperplasia and decreased arterial SHP-1 expression compared with mice fed a regular diet. Arterial SHP-1 expression was also decreased in Zucker fatty rats, Zucker diabetic fatty rats and in patients with type 2 diabetes. In primary cultured VSMCs, oxidised LDL suppressed SHP-1 expression by activating Mek-1 (also known as Map2k1) and increased DNA methylation of the Shp-1 promoter. VSMCs from Shp-1-Tg mice exhibited impaired platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation with a concomitant decrease in PDGF-stimulated VSMC proliferation and migration. Similarly, HFD-fed Shp-1-Tg mice and mice treated with the SHP-1 inducer, Icariside II, were protected from the development of intimal hyperplasia following wire injury. CONCLUSIONS/INTERPRETATION: Suppression of SHP-1 by oxidised lipids may contribute to the excessive VSMC proliferation, inflammatory cytokine production and intimal hyperplasia observed in arteries from diabetes and insulin resistance. Augmenting SHP-1 levels is a potential therapeutic strategy to maintain stent patency in patients with insulin resistance and diabetes.

Revascularization and muscle adaptation to limb demand ischemia in diet-induced obese mice.

BACKGROUND: Obesity and type 2 diabetes are major risk factors for peripheral arterial disease in humans, which can result in lower limb demand ischemia and exercise intolerance. Exercise triggers skeletal muscle adaptation including increased vasculogenesis. The goal of this study was to determine whether demand ischemia modulates revascularization, fiber size, and signaling pathways in the ischemic hind limb muscles of mice with diet-induced obesity (DIO). MATERIALS AND METHODS: DIO mice (n = 7) underwent unilateral femoral artery ligation and recovered for 2 wks followed by 4 wks with daily treadmill exercise to induce demand ischemia. A parallel sedentary ischemia (SI) group (n = 7) had femoral artery ligation without exercise. The contralateral limb muscles of SI served as control. Muscles were examined for capillary density, myofiber cross-sectional area, cytokine levels, and phosphorylation of STAT3 and ERK1/2. RESULTS: Exercise significantly enhanced capillary density (P < 0.01) and markedly lowered cross-sectional area (P < 0.001) in demand ischemia compared with SI. These findings coincided with a significant increase in granulocyte colony-stimulating factor (P < 0.001) and interleukin-7 (P < 0.01) levels. In addition, phosphorylation levels of STAT3 and ERK1/2 (P < 0.01) were increased, whereas UCP1 and monocyte chemoattractant protein-1 protein levels were lower (P < 0.05) without altering vascular endothelial growth factor and tumor necrosis factor alpha protein levels. Demand ischemia increased the PGC1α messenger RNA (P < 0.001) without augmenting PGC1α protein levels. CONCLUSIONS: Exercise-induced limb demand ischemia in the setting of DIO causes myofiber atrophy despite an increase in muscle capillary density. The combination of persistent increase in tumor necrosis factor alpha, lower vascular endothelial growth factor, and failure to increase PGC1α protein may reflect a deficient adaption to demand ischemia in DIO.

Insulin Downregulates the Transcriptional Coregulator CITED2, an Inhibitor of Proangiogenic Function in Endothelial Cells.

In patients with atherosclerotic complications of diabetes, impaired neovascularization of ischemic tissue in the myocardium and lower limb limits the ability of these tissues to compensate for poor perfusion. We identified 10 novel insulin-regulated genes, among them Adm, Cited2, and Ctgf, which were downregulated in endothelial cells by insulin through FoxO1. CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which was downregulated by insulin by up to 54%, is an important negative regulator of hypoxia-inducible factor (HIF) and impaired HIF signaling is a key mechanism underlying the impairment of angiogenesis in diabetes. Consistent with impairment of vascular insulin action, CITED2 was increased in cardiac endothelial cells from mice with diet-induced obesity and from db/db mice and was 3.8-fold higher in arterial tissue from patients with type 2 diabetes than control subjects without diabetes. CITED2 knockdown promoted endothelial tube formation and endothelial cell proliferation, whereas CITED2 overexpression impaired HIF activity in vitro. After femoral artery ligation, induction of an endothelial-specific HIF target gene in hind limb muscle was markedly upregulated in mice with endothelial cell deletion of CITED2, suggesting that CITED2 can limit HIF activity in vivo. We conclude that vascular insulin resistance in type 2 diabetes contributes to the upregulation of CITED2, which impairs HIF signaling and endothelial proangiogenic function.

Insulin decreases atherosclerosis by inducing endothelin receptor B expression.

Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe-/- mice (Irs1/Apoe-/-) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE-/- mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin's enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE-/- mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr-/- and Irs1/Ldlr-/- mice decreased NO production and accelerated atherosclerosis, compared with Ldlr-/- mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production.

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 PKCß2 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 PKCß2-dominant negative or selective PKCß 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 PKCß 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.

Induction of vascular insulin resistance and endothelin-1 expression and acceleration of atherosclerosis by the overexpression of protein kinase C-ß isoform in the endothelium.

RATIONALE: Loss of insulin action in the endothelium can cause endothelial dysfunction and atherosclerosis. Hyperglycemia and elevated fatty acids induced by diabetes mellitus can activate protein kinase C-ß isoforms and selectively inhibit insulin signaling via phosphatidylinositol 3-kinase/Akt pathway to inhibit the activation of endothelial nitric oxide synthase and metabolic actions. OBJECTIVE: To demonstrate that overexpressing protein kinase C-ß2 isoform in endothelial cells can cause selective insulin resistance and exacerbate atherosclerosis in the aorta. METHODS AND RESULTS: Protein kinase C-ß2 isoform was overexpressed in endothelial cells using a promoter of vascular endothelial cell cadherin. These mice were cross-bred with apoE-/- mice [Tg (Prkcb)apoE-/-]. On a Western diet, Tg(Prkcb)apoE-/- and apoE-/- mice did not differ in systemic insulin sensitivity, glucose tolerance, plasma lipid, or blood pressure. Insulin action in endothelial cells and femoral artery from Tg(Prkcb)apoE-/- mice was impaired by ≈40% with respect to Akt/endothelial nitric oxide synthase activation, and leukocyte-endothelial cell binding increased in cultured lung endothelial cells from Tg(Prkcb)apoE-/- mice compared with that from apoE-/- mice. Basal and angiotensin-stimulated big endothelin-1 levels were elevated in Tg(Prkcb)apoE-/- mice compared with apoE-/- mice. The severity of atherosclerosis in the aorta from Tg(Prkcb)apoE-/- mice increased by ≈70% as measured by en face fat staining and plaque content of the number of smooth muscle cells, macrophages, and extracellular matrix. CONCLUSIONS: Specific protein kinase C-ß2 activation in the endothelial cells caused dysfunction and accelerated atherosclerosis because of loss of insulin-stimulated Akt/endothelial nitric oxide synthase activation and angiotensin-induced increases in endothelin-1 expression.

Vascular complications of diabetes: mechanisms of injury and protective factors.

In patients with diabetes, atherosclerosis is the main reason for impaired life expectancy, and diabetic nephropathy and retinopathy are the largest contributors to end-stage renal disease and blindness, respectively. An improved therapeutic approach to combat diabetic vascular complications might include blocking mechanisms of injury as well as promoting protective or regenerating factors, for example by enhancing the action of insulin-regulated genes in endothelial cells, promoting gene programs leading to induction of antioxidant or anti-inflammatory factors, or improving the sensitivity to vascular cell survival factors. Such strategies could help prevent complications despite suboptimal metabolic control.

Retinal not systemic oxidative and inflammatory stress correlated with VEGF expression in rodent models of insulin resistance and diabetes.

PURPOSE: To correlate changes between VEGF expression with systemic and retinal oxidative stress and inflammation in rodent models of obesity induced insulin resistance and diabetes. METHODS: Retinal VEGF mRNA and protein levels were assessed by RT-PCR and VEGF ELISA, respectively. Urinary 8-hydroxydeoxyguanosine (8-OHdG), blood levels of C-reactive protein (CRP), malondialdehyde (MDA), and CD11b/c positive cell ratio were used as systemic inflammatory markers. Retinal expression of Nox2, Nox4, and p47phox mRNA levels were measured as oxidative stress markers. TNF-α, inter-cellular adhesion molecule-1 (ICAM-1), IL1ß, and activation of nuclear factor κB (NF-κB) were used as retinal inflammatory markers. RESULTS: Retinal VEGF mRNA and protein expression increased in Zucker diabetic fatty (ZDF(fa/fa)) rats and streptozotosin (STZ) induced diabetic Sprague-Dawley rats, after two months of disease, but not in Zucker fatty (ZF) rats. Systemic markers of oxidative stress and inflammation were elevated in insulin resistant and diabetic rats. Some oxidative stress and inflammatory markers (TNF-α, IL-6, ICAM-1, and IL1-ß) were upregulated in the retina of ZDF(fa/fa) and STZ diabetic rats after 4 months of disease. In contrast, activation of NF-κB in the retina was observed in high fat fed nondiabetic and diabetic cis-NF-κB(EGFP) mice, ZF, ZDF(fa/fa), and STZ-induced diabetic rats. CONCLUSIONS: Only persistent hyperglycemia and diabetes increased retinal VEGF expression. Some markers of inflammation and oxidative stress were elevated in the retina and systemic circulation of obese and insulin resistant rodents with and without diabetes. Induction of VEGF and its associated retinal pathologies by diabetes requires chronic hyperglycemia and factors in addition to inflammation and oxidative stress.

Tissue-specific insulin signaling, metabolic syndrome, and cardiovascular disease.

Impaired insulin signaling is central to development of the metabolic syndrome and can promote cardiovascular disease indirectly through development of abnormal glucose and lipid metabolism, hypertension, and a proinflammatory state. However, insulin's action directly on vascular endothelium, atherosclerotic plaque macrophages, and in the heart, kidney, and retina has now been described, and impaired insulin signaling in these locations can alter progression of cardiovascular disease in the metabolic syndrome and affect development of microvascular complications of diabetes mellitus. Recent advances in our understanding of the complex pathophysiology of insulin's effects on vascular tissues offer new opportunities for preventing these cardiovascular disorders.

Protective effects of GLP-1 on glomerular endothelium and its inhibition by PKCß activation in diabetes.

To characterize glucagon-like peptide (GLP)-1 signaling and its effect on renal endothelial dysfunction and glomerulopathy. We studied the expression and signaling of GLP-1 receptor (GLP-1R) on glomerular endothelial cells and the novel finding of protein kinase A-dependent phosphorylation of c-Raf at Ser259 and its inhibition of angiotensin II (Ang II) phospho-c-Raf(Ser338) and Erk1/2 phosphorylation. Mice overexpressing protein kinase C (PKC)ß2 in endothelial cells (EC-PKCß2Tg) were established. Ang II and GLP-1 actions in glomerular endothelial cells were analyzed with small interfering RNA of GLP-1R. PKCß isoform activation induced by diabetes decreased GLP-1R expression and protective action on the renal endothelium by increasing its degradation via ubiquitination and enhancing phospho-c-Raf(Ser338) and Ang II activation of phospho-Erk1/2. EC-PKCß2Tg mice exhibited decreased GLP-1R expression and increased phospho-c-Raf(Ser338), leading to enhanced effects of Ang II. Diabetic EC-PKCß2Tg mice exhibited greater loss of endothelial GLP-1R expression and exendin-4-protective actions and exhibited more albuminuria and mesangial expansion than diabetic controls. These results showed that the renal protective effects of GLP-1 were mediated via the inhibition of Ang II actions on cRaf(Ser259) and diminished by diabetes because of PKCß activation and the increased degradation of GLP-1R in the glomerular endothelial cells.

Glomerular VEGF resistance induced by PKCδ/SHP-1 activation and contribution to diabetic nephropathy.

This study characterizes the effect of glucose-induced activation of protein kinase Cδ (PKCδ) and Src homology-2 domain-containing phosphatase-1 (SHP-1) expression on vascular endothelial growth factor (VEGF) actions in glomerular podocytes in cultures and in glomeruli of diabetic rodents. Elevation of glucose levels induced PKCδ and p38 mitogen-activated protein kinase (p38 MAPK) to increase SHP-1 expression, increased podocyte apoptosis, and inhibited VEGF activation in podocytes and glomerular endothelial cells. The adverse effects of high glucose levels can be negated by molecular inhibitors of PKCδ, p38MAPK, and SHP-1 and only partially reduced by antioxidants and nuclear factor-κB (NF-κB) inhibitor. Increased PKCδ activation and SHP-1 expression correlated with loss of VEGF signaling and podocyte numbers in the glomeruli of diabetic rats and mice. In contrast, diabetic PKCδ-knockout (Prkcd(-/-)) mice did not exhibit activation of p38 MAPK and SHP-1 or inhibition of VEGF signaling in renal glomeruli. Functionally, diabetic Prkcd(-/-) mice had decreased expressions of TGFß, VEGF, and extracellular matrix and less albuminuria than diabetic Prkcd(+/+) mice. Hyperglycemia and diabetes can cause glomerular podocyte apoptosis and endothelial dysfunction partly due to increased PKCδ/p38 MAPK activation and the expression of SHP-1 to cause VEGF resistance, independent of NF-κB activation.