Kalyankar Mahadev

The NAD(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation of H2O2 and Plays an Integral Role in Insulin Signal Transduction

ABSTRACT Insulin stimulation of target cells elicits a burst of H2O2 that enhances tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins as well as distal signaling events in the insulin action cascade. The molecular mechanism coupling the insulin receptor with the cellular oxidant-generating apparatus has not been elucidated. Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells. Adenovirus-mediated expression of Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains acted in a dominant-negative fashion in differentiated 3T3-L1 adipocytes and attenuated insulin-stimulated H2O2 generation, insulin receptor (IR) and IRS-1 tyrosine phosphorylation, activation of downstream serine kinases, and glucose uptake. Transfection of specific small interfering RNA oligonucleotides reduced Nox4 protein abundance and also inhibited the insulin signaling cascade. Overexpression of Nox4 also significantly reversed the inhibition of insulin-stimulated IR tyrosine phosphorylation induced by coexpression of PTP1B by inhibiting PTP1B catalytic activity. These data suggest that Nox4 provides a novel link between the IR and the generation of cellular reactive oxygen species that enhance insulin signal transduction, at least in part via the oxidative inhibition of cellular protein-tyrosine phosphatases (PTPases), including PTP1B, a PTPase that has been previously implicated in the regulation of insulin action.

Adiponectin deficiency increases leukocyte-endothelium interactions via upregulation of endothelial cell adhesion molecules in vivo

This study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad(-/-)) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad(-/-) mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad(-/-) mice significantly attenuated leukocyte-endothelium interactions and adhesion molecule expression in addition to restoring physiologic levels of eNO. Importantly, prior administration of gAd also protected WT mice against TNF-alpha-induced leukocyte-endothelium interactions, indicating a pharmacologic action of gAd. Mechanistically, blockade of eNOS with N(omega)-nitro-L-arginine methyl ester ( L-NAME) abolished the inhibitory effect of gAd on leukocyte adhesion, demonstrating the obligatory role of eNOS signaling in the antiinflammatory action of gAd. We believe this is the first demonstration that gAd protects the vasculature in vivo via increased NO bioavailability with suppression of leukocyte-endothelium interactions. Overall, we provide evidence that loss of adiponectin induces a primary state of endothelial dysfunction with increased leukocyte-endothelium adhesiveness.

Adiponectin Suppression of High-Glucose–Induced Reactive Oxygen Species in Vascular Endothelial Cells Evidence for Involvement of a cAMP Signaling Pathway

Adiponectin is an abundant adipocyte-derived plasma protein with antiatherosclerotic effects. Vascular signal transduction by adiponectin is poorly understood and may involve 5′-AMP–activated protein kinase (AMPK), cAMP signaling, and other pathways. Hyperglycemia sharply increases the production of reactive oxygen species (ROS), which play a key role in endothelial dysfunction in diabetes. Because the recombinant globular domain of human adiponectin (gAd) reduces the generation of endothelial ROS induced by oxidized LDL, we sought to determine whether adiponectin could also suppress ROS production induced by high glucose in cultured human umbilical vein endothelial cells. Incubation in 25 mmol/l glucose for 16 h increased ROS production 3.8-fold (P < 0.05), using a luminol assay. Treatment with gAd for 16 h suppressed glucose-induced ROS in a dose-dependent manner up to 81% at 300 nmol/l (P < 0.05). The AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 1 mmol/l, 16 h) only partially decreased glucose-induced ROS by 22% (P < 0.05). Cell pretreatment with AMPK inhibitors, however, failed to block the effect of gAd to suppress glucose-induced ROS, suggesting that the action of gAd was independent of AMPK. Interestingly, activation of cAMP signaling by treatment with forskolin (2 μmol/l) or dibutyryl-cAMP (0.5 mmol/l) reduced glucose-induced ROS generation by 43 and 67%, respectively (both P < 0.05). Incubation with the cAMP-dependent protein kinase (PKA) inhibitor H-89 (1 μmol/l) fully abrogated the effect of gAd, but not that of AICAR, on ROS induced by glucose. gAd also increased cellular cAMP content by 70% in an AMPK-independent manner. Full-length adiponectin purified from a eukaryotic expression system also suppressed ROS induced by high glucose or by treatment of endothelial cells with oxidized LDL. Thus, adiponectin suppresses excess ROS production under high-glucose conditions via a cAMP/PKA-dependent pathway, an effect that has implications for vascular protection in diabetes.

Role of Protein Tyrosine Phosphatase 1B in Vascular Endothelial Growth Factor Signaling and Cell–Cell Adhesions in Endothelial Cells

Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin–mediated cell–cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin–mediated cell–cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell–cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.

Inhibition of Reactive Oxygen Species by Lovastatin Downregulates Vascular Endothelial Growth Factor Expression and Ameliorates Blood-Retinal Barrier Breakdown in db/db Mice: Role of NADPH Oxidase 4

OBJECTIVE Oxidative stress is a key pathogenic factor in diabetic retinopathy. We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in diabetic retinopathy. RESEARCH DESIGN AND METHODS Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α; production of reactive oxygen species (ROS); and retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCECs) and in db/db mice treated with lovastatin. RESULTS Expressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCECs, hypoxia and high glucose upregulated mRNA and protein expression of Nox4, ROS generation, and VEGF level. These changes were abrogated by pretreatment with lovastatin or NADPH oxidase inhibitor diphenyleneiodonium chloride. Overexpression of Nox4 increased basal level of ROS generation, HIF-1α, and VEGF expression in RCECs. In contrast, blockade of Nox4 activity using adenovirus-expressing dominant-negative Nox4 abolished hypoxia- and high-glucose–induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1α and high-glucose–elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 small interfering RNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice. CONCLUSIONS Activation of Nox4 plays an important role in high-glucose– and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4, at least in part, contributes to the protective effects of lovastatin in diabetic retinopathy.

Inhibition of reactive oxygen species by lovastatin down-regulates VEGF expression and ameliorates blood-retinal barrier breakdown in db/db mice: role of NADPH oxidase 4

OBJECTIVE Oxidative stress is a key pathogenic factor in diabetic retinopathy. We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in diabetic retinopathy. RESEARCH DESIGN AND METHODS Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α; production of reactive oxygen species (ROS); and retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCECs) and in db/db mice treated with lovastatin. RESULTS Expressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCECs, hypoxia and high glucose upregulated mRNA and protein expression of Nox4, ROS generation, and VEGF level. These changes were abrogated by pretreatment with lovastatin or NADPH oxidase inhibitor diphenyleneiodonium chloride. Overexpression of Nox4 increased basal level of ROS generation, HIF-1α, and VEGF expression in RCECs. In contrast, blockade of Nox4 activity using adenovirus-expressing dominant-negative Nox4 abolished hypoxia- and high-glucose–induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1α and high-glucose–elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 small interfering RNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice. CONCLUSIONS Activation of Nox4 plays an important role in high-glucose– and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4, at least in part, contributes to the protective effects of lovastatin in diabetic retinopathy.

Adiponectin Protects Against Angiotensin II or Tumor Necrosis Factor α–Induced Endothelial Cell Monolayer Hyperpermeability Role of cAMP/PKA Signaling

Objective—Angiotensin II (Ang II) and tumor necrosis factor (TNF)-&agr; levels increase endothelial permeability, and we hypothesized that adiponectin suppressed these responses in a cAMP-dependent manner. Methods and Results—The effect of adiponectin on transendothelial electric resistance (TEER) and diffusion of albumin through human umbilical vein and bovine aortic endothelial cell monolayers induced by Ang II (100 nmol/L) or TNF-&agr; (5 ng/mL) was measured. Treatment with the globular domain of adiponectin (3 &mgr;g/mL) for 16 hours abrogated the adverse TEER effect of TNF-&agr; (−35 versus −12 &OHgr;/cm2 at 45 minutes, P<0.05) and Ang II (−25 versus −5 &OHgr;/cm2 at 45 minutes, P<0.01) and partially suppressed the increased diffusion of albumin with Ang II (40% versus 10% change, P<0.05) or TNF-&agr; (40% versus 20% change, P<0.05). Full-length adiponectin also suppressed Ang II–induced monolayer hyperpermeability. Adiponectin treatment also suppressed Ang II–induced increased actin stress fiber development, intercellular gap formation, and &bgr;-tubulin disassembly. Adiponectin increased cAMP levels, and its effects were abrogated by inhibition of adenylyl cyclase or cAMP-dependent protein kinase signaling. Conclusions—Adiponectin protects the endothelial monolayer from Ang II or TNF-&agr;-induced hyperpermeability by modulating microtubule and cytoskeleton stability via a cAMP/ PKA signaling cascade.

Protein-tyrosine phosphatase activity in human adipocytes is strongly correlated with insulin-stimulated glucose uptake and is a target of insulin-induced oxidative inhibition.

Protein-tyrosine phosphatases (PTPases), in particular PTP1B, have been shown to modulate insulin signal transduction in liver and skeletal muscle in animal models; however, their role in human adipose tissue remains unclear. The uptake of (14)C-D-glucose in response to 10 or 100 nmol/L insulin was measured in isolated subcutaneous adipocytes from subjects with a mean age of 44 years (range, 26 to 58) and mean body mass index (BMI) of 35.6 (range, 29.7 to 45.5). The endogenous activity of total PTPases and specifically of PTP1B in immunoprecipitates was measured in cell lysates under an inert atmosphere with and without added reducing agents. Using nonlinear regression analysis, higher BMI was significantly correlated with lower adipocyte glucose uptake (r = 0.73, P =.01) and with increased endogenous total PTPase activity (r = 0.64, P =.04). Correlation with waist circumference gave similar results. The endogenous total PTPase activity also strongly correlated with insulin-stimulated glucose uptake (R =.89, P <.0001); however, the activity of PTP1B was unrelated to the level of glucose uptake. Consistent with the insulin-stimulated oxidative inhibition of thiol-dependent PTPases reported for 3T3-L1 adipocytes and hepatoma cells, treatment of human adipocytes with 100 nmol/L insulin for 5 minutes lowered endogenous PTPase activity to 37% of control (P <.001), which was increased 25% by subsequent treatment with dithiothreitol in vitro. Cellular treatment with diphenyleneiodonium (DPI), an NADPH oxidase inhibitor that blocks the cellular generation of H(2)O(2) and reduces the insulin-induced reduction of cellular PTPase activity, also diminished insulin-stimulated glucose uptake by 82% (P =.001). These data suggest that total cellular PTPase activity, but not the activity of PTP1B, is higher in more obese subjects and is negatively associated with insulin-stimulated glucose transport. The insulin-stimulated oxidative inhibition of PTPases may also have an important permissive role in the transmission of the insulin signal to glucose transport in human adipocytes.

Use of an anaerobic environment to preserve the endogenous activity of protein-tyrosine phosphatases isolated from intact cells

Protein‐tyrosine phosphatases (PTPases) have a common cysteine residue whose reduced state is integral to their phosphocysteine‐mediated reaction mechanism. The catalytic cysteine thiol can be oxidized or conjugated during cellular redox reactions, which provides an important means of PTPase regulation in vivo. Because exposure to air can artifactually oxidize this reactive thiol, PTPase assays have typically used potent reducing agents such as dithiothreitol to reactivate the enzymes present. However, this approach does not allow for the measurement of endogenous PTPase activity as directly isolated from the in vivo cellular environment. Here we show that sample processing and assay in an anaerobic chamber by using deoxygenated buffers can preserve the overall activity of PTPases in subcellular fractions of 3T3‐L1 adipocytes, HepG2 hepatoma cells, and human adipose tissue, as well as with PTP1B, specifically isolated by immunoprecipitation. Cell lysis into air reduced the PTPase activity to as low as 20% of the level observed with sample handling in the anaerobic environment, which was variably restored towards the activity in the anaerobic samples by treatment with dithiothreitol. The approach reported here provides a new framework for characterizing the activity of PTPases as isolated from the intracellular milieu, which more closely reflects the endogenous reactivity and potential impact of these PTPases on signal transduction pathways involving reversible protein‐tyrosine phosphorylation.

Comment on: Hattori et al. (2007) Globular Adiponectin Activates Nuclear Factor-κB and Activating Protein-1 and Enhances Angiotensin II–Induced Proliferation in Cardiac Fibroblasts: Diabetes 56:804–808

We read with interest the recent articles by Hattori et al. (1,2), which report activation of nuclear factor-κB (NF-κB) transcription by the recombinant globular domain of adiponectin. However, these findings contrast directly with a substantial body of published and ongoing work. As initially reported by Ouchi et al. and confirmed by other groups (3,4), a major effect of adiponectin is the suppression of proinflammatory endothelial responses, which include reversing the activation of NF-κB, reducing adhesion molecule expression, and enhancing nitric oxide bioavailability. We have shown that the globular domain of adiponectin also suppresses endothelial reactive oxygen species generation in response to various agonists, including oxidized LDL and high glucose (5,6). At the American Diabetes Association 66th …