Diabetes is a high risk factor for dementia. High glucose may be a risk factor for dementia even among persons without diabetes, and in transgenic animals it has been shown to cause a potentiation of indices that are pre-symptomatic of Alzheimer's disease. To further elucidate the underlying mechanisms linking inflammatory events elicited in the brain during oxidative stress and diabetes, we monitored the activation of mitogen-activated kinsase (MAPKs), c-jun NH2-terminal kinase (JNK), p38 MAP kinases (p38MAPK), and extracellular activating kinsae1/2 (ERK1/2) and the anti-inflammatory effects of the thioredoxin mimetic (TxM) peptides, Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4) in the brain of male leptin-receptor-deficient Zucker diabetic fatty (ZDF) rats and human neuroblastoma SH-SY5Y cells. Daily i.p. injection of CB3 to ZDF rats inhibited the phosphorylation of JNK and p38MAPK, and prevented the expression of thioredoxin-interacting-protein (TXNIP/TBP-2) in ZDF rat brain. Although plasma glucose/insulin remained high, CB3 also increased the phosphorylation of AMP-ribose activating kinase (AMPK) and inhibited p70S6K kinase in the brain. Both CB3 and CB4 reversed apoptosis induced by inhibiting thioredoxin reductase as monitored by decreasing caspase 3 cleavage and PARP dissociation in SH-SY5Y cells. The decrease in JNK and p38MAPK activity in the absence of a change in plasma glucose implies a decrease in oxidative or neuroinflammatory stress in the ZDF rat brain. CB3 not only attenuated MAPK phosphorylation and activated AMPK in the brain, but it also diminished apoptotic markers, most likely acting via the MAPK–AMPK–mTOR pathway. These results were correlated with CB3 and CB4 inhibiting inflammation progression and protection from oxidative stress induced apoptosis in human neuronal cells. We suggest that by attenuating neuro-inflammatory processes in the brain Trx1 mimetic peptides could become beneficial for preventing neurological disorders associated with diabetes.
•Thioredoxin mimeitics peptides (TXM) lower apoptosis in the brain of ZDF rat.•TxM peptides prevent TXNIP/TBP-2 expression in the brain of ZDF rat.•TxM peptides could become beneficial for preventing diabetes associated neurological disorders.
Ad-AMPK-CA, AMPK-constitutively active AMP-activated protein kinase mutants; AICAR, 5-amino-4-imidazole carboxamide riboside; AMPK, AMP-activated protein kinase; TXNIP/TBP-2, thioredoxin-interacting protein; CB3, NAc-Cys-Pro Cys-amide, TXM-CB3; Diabetes type 2; Inflammation; Thioredoxin mimetics; ZDF rat-model; MAPK; AMPK; TXNIP/TBP-2; CB3; Oxidative stress; Redox
NADPH oxidase–generated reactive oxygen species (ROS) are implicated in angiogenesis. Isoforms of NADPH oxidase NOX1, NOX2, and NOX4 are reported to be expressed in endothelial cells (ECs). Of these, NOX1 and NOX2 have been reported to contribute to intravitreal neovascularization (IVNV) in oxygen-induced retinopathy (OIR) models. In this study, we tested the hypothesis that the isoform NOX4 in ECs contributed to vascular endothelial growth factor (VEGF)–induced angiogenesis and IVNV.
Isoforms of NADPH oxidase mRNA were measured in several types of cultured vascular ECs: human retinal microvascular ECs (hRMVECs), choroidal ECs (CECs), and human umbilical vascular ECs (HUVECs) using real-time PCR. Newborn rat pups and dams were placed into an OIR model that cycled oxygen concentration between 50% and 10% every 24 h for 14 days, and then were placed in room air (RA) for an additional 4 days (rat OIR model). NOX4 expression in retinal lysates from the RA–raised pups at postnatal day 0 (P0), P14, and P18 was determined with western blots. STAT3 activation was determined as the ratio of phosphorylated STAT3 to total STAT3 with western blot analysis of retinal lysates from pups raised in RA or from the rat OIR model at P18. Semiquantitative assessment of the density of NOX4 colabeling with lectin-stained retinal ECs was determined by immunolabeling of retinal cryosections from P18 pups in OIR or in RA. In hRMVECs transfected with NOX4 siRNA and treated with VEGF or control, 1) ROS generation was measured using the 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester fluorescence assay and 2) phosphorylated VEGF receptor 2 and STAT3, and total VEGFR2 and STAT3 were measured in western blot analyses. VEGF-stimulated hRMVEC proliferation was measured following transfection with NOX4 siRNA or STAT3 siRNA, or respective controls.
NOX4 was the most prevalent isoform of NADPH oxidase in vascular ECs. NOX4 expression in retinal lysates was significantly decreased during development in RA. Compared to RA, the expression of retinal NOX4 increased at P18. At p18 OIR, semiquantitative assessment of the density of lectin and NOX4 colabeling in retinal vascular ECs was greater in retinal cryosections and activated STAT3 was greater in retinal lysates when compared to the RA-raised pups. In cultured hRMVECs, knockdown of NOX4 by siRNA transfection inhibited VEGF-induced ROS generation. VEGF induced a physical interaction of phosphorylated-VEGFR2 and NOX4. Knockdown of NOX4: 1) reduced VEGFR2 activation but did not abolish it and 2) abolished STAT3 activation in response to VEGF. Knockdown of either NOX4 or STAT3 inhibited VEGF-induced EC proliferation.
Our data suggest that in a model representative of human retinopathy of prematurity, NOX4 was increased at a time point when IVNV developed. VEGF-activated NOX4 led to an interaction between VEGF-activated VEGFR2 and NOX4 that mediated EC proliferation via activation of STAT3. Altogether, our results suggest that NOX4 may regulate VEGFR2-mediated IVNV through activated STAT3.
Although Nox5, (Nox2 homologue), has been identified in the vasculature, its regulation and functional significance remain unclear.
To test if vasoactive agents regulate Nox5 through Ca2+/calmodulin-dependent processes and whether Ca2+ -sensitive Nox5, associated with Rac-1, generates superoxide (•O2−) and activates growth and inflammatory responses via MAP kinases in human endothelial cells (ECs).
Methods and results
Cultured ECs, exposed to AngII and ET-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor) and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with siRNA. AngII and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated •O2− production and inhibited phosphorylation of ERK1/2, but not p38MAPK or SAPK/JNK. Nox5 siRNA blunted AngII-induced, but not ET-1-induced, upregulation of PCNA and VCAM-1, important in growth and inflammation.
Human ECs possess functionally active Nox5, regulated by AngII and ET-1 through Ca2+/calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by AngII and ET-1 induces ROS generation and ERK 1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by AngII but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by AngII and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by AngII and ET-1.
Reactive oxygen species; vascular cells; vasoactive peptides; ERK1/2
The objective of this study was to determine whether ER stress correlates with β-cell dysfunction in obesity-associated diabetes.
Quantitative RT-PCR and western blot analysis were used to investigate changes in the expression of markers of ER stress, the unfolded protein response (UPR) and β-cell function in islets isolated from (1) non-diabetic Zucker obese (ZO) and obese female Zucker diabetic fatty (fZDF) rats compared with their lean littermates and from (2) high-fat-diet-fed fZDF rats (HF-fZDF), to induce diabetes, compared with age-matched non-diabetic obese fZDF rats.
Markers of an adaptive ER stress/UPR and β-cell function are elevated in islets isolated from ZO and fZDF rats compared with their lean littermates. In islets isolated from HF-fZDF rats, there was no significant change in the expression of markers of ER stress compared with age matched, obese, non-diabetic fZDF rats.
These results provide evidence that obesity-induced activation of the UPR is an adaptive response for increasing the ER folding capacity to meet the increased demand for insulin. As ER stress is not exacerbated in high-fat-diet-induced diabetes, we suggest that failure of the islet to mount an effective adaptive UPR in response to an additional increase in insulin demand, rather than chronic ER stress, may ultimately lead to β-cell failure and hence diabetes.
ER stress; unfolded protein response; obesity; pancreatic β-cell; type-2 diabetes
Glucose-stimulated insulin secretion [GSIS] involves a sequence of metabolic events leading to small G-protein [e.g., Rac1]-mediated cytoskeletal remodeling to promote granule mobilization toward the plasma membrane for fusion and release of insulin. Existing evidence supports a positive modulatory role for Rac1 in GSIS. Specific regulatory factors of Rac1 function, including the guanine nucleotide exchange factors [e.g., Tiam1] have also been identified and studied in the islet. Inhibition of Tiam1/Rac1 signaling axis attenuates GSIS suggesting its pivotal role in insulin secretion. In addition to its positive [i.e., friendly] roles in GSIS, Rac1 also plays “non-friendly” role[s] in the islet function. For example, it up-regulates the intracellular reactive oxygen species [ROS] levels via activation of phagocyte-like NADPH oxidase [Nox]. Despite the emerging evidence that a tonic increase in intracellular ROS is necessary for GSIS, experimental evidence also suggests that chronic exposure of β-cells to high glucose, palmitate or cytokines results in the onset of oxidative stress leading to reduction in mitochondrial membrane potential, cytosolic accumulation of cytochrome C and activation of caspase-3 leading to β-cell apoptosis. Pharmacological and molecular biological inhibition of Rac1 activation affords partial protection against Nox-induced oxidative stress and mitochondrial dysfunction induced by elevated glucose, lipids or cytokines. Herein, we overview the existing evidence to suggest positive as well as negative modulatory roles of Rac1 in islet function. Potential avenues for future research including development of inhibitors to halt the Rac1-Nox activation and generation of oxidative stress leading to the metabolic dysfunction of the β-cell are discussed.
Rac1; Tiam1; NADPH-oxidase; Pancreatic islet; mitochondrial dysfunction and insulin secretion
Endothelial dysfunction in resistance arteries alters end organ perfusion in type 2 diabetes. Superoxides and cyclooxygenase-2 (COX-2) derivatives have been shown separately to alter endothelium-mediated relaxation in aging and diabetes but their role in the alteration of vascular tone in old diabetic subjects is not clear, especially in resistance arteries. Consequently, we investigated the role of superoxide and COX-2-derivatives on endothelium-dependent relaxation in 3 and 12 month-old Zucker diabetic fatty (ZDF) and lean (LZ) rats. Mesenteric resistance arteries were isolated and vascular tone was investigated using wire-myography. Endothelium (acetylcholine)-dependent relaxation was lower in ZDF than in LZ rats (60 versus 84% maximal relaxation in young rats and 41 versus 69% in old rats). Blocking NO production with L-NAME was less efficient in old than in young rats. L-NAME had no effect in old ZDF rats although eNOS expression level in old ZDF rats was similar to that in old LZ rats. Superoxide level and NADPH-oxidase subunits (p67phox and gp91phox) expression level were greater in ZDF than in LZ rats and were further increased by aging in ZDF rats. In young ZDF rats reducing superoxide level with tempol restored acetylcholine-dependent relaxation to the level of LZ rats. In old ZDF rats tempol improved acetylcholine-dependent relaxation without increasing it to the level of LZ rats. COX-2 (immunolabelling and Western-blot) was present in arteries of ZDF rats and absent in LZ rats. In old ZDF rats arterial COX-2 level was higher than in young ZDF rats. COX-2 blockade with NS398 restored in part acetylcholine-dependent relaxation in arteries of old ZDF rats and the combination of tempol and NS398 fully restored relaxation in control (LZ rats) level. Accordingly, superoxide production and COX-2 derivatives together reduced endothelium-dependent relaxation in old ZDF rats whereas superoxides alone attenuated relaxation in young ZDF or old LZ rats.
The phagocytic NADPH-oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.
NADPH Oxidase; Rac1; Tiam1; palmitate; ceramide; oxidative stress; pancreatic β-cells
The Zucker diabetic fatty (ZDF) rat is a genetic model in which the homozygous (FA/FA) male animals develop obesity and type 2 diabetes. Morbidity and mortality from cardiovascular complications, due to increased oxidative stress and inflammatory signals, are the hallmarks of type 2 diabetes. The precise molecular mechanism of contractile dysfunction and disease progression remains to be clarified. Therefore, we have investigated molecular and metabolic targets in male ZDF (30–34 weeks old) rat heart compared to age matched Zucker lean (ZL) controls. Hyperglycemia was confirmed by a 4-fold elevation in non-fasting blood glucose (478.43 ± 29.22 mg/dL in ZDF vs. 108.22 ± 2.52 mg/dL in ZL rats). An increase in reactive oxygen species production, lipid peroxidation and oxidative protein carbonylation was observed in ZDF rats. A significant increase in CYP4502E1 activity accompanied by increased protein expression was also observed in diabetic rat heart. Increased expression of other oxidative stress marker proteins, HO-1 and iNOS was also observed. GSH concentration and activities of GSH-dependent enzymes, glutathione S-transferase and GSH reductase, were, however, significantly increased in ZDF heart tissue suggesting a compensatory defense mechanism. The activities of mitochondrial respiratory enzymes, Complex I and Complex IV were significantly reduced in the heart ventricle of ZDF rats in comparison to ZL rats. Western blot analysis has also suggested a decreased expression of IκB-α and phosphorylated-JNK in diabetic heart tissue. Our results have suggested that mitochondrial dysfunction and increased oxidative stress in ZDF rats might be associated, at least in part, with altered NF-κB/JNK dependent redox cell signaling. These results might have implications in the elucidation of the mechanism of disease progression and designing strategies for diabetes prevention.
Zucker rats; diabetes; obesity; oxidative stress; cardiomyocytes; mitochondria
Background & Aims
NADPH oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex including Rac1. Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis.
To induce liver fibrosis, wild-type (WT) and SOD1mu mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL). Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of TBARS and NOX related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knock-out (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression.
Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor GKT137831 attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as mRNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and TGFb upregulated NOX4, but AngII required NOX1.
SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 upregulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis.
NADPH oxidase; SOD1; reactive oxidative species; hepatic stellate cells
Aim: Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinson's disease. However, molecular sources for reactive oxygen species in Parkinson's disease have not been clearly elucidated. Herein, we sought to investigate whether a superoxide-producing NADPH oxidases (NOXs) are implicated in oxidative stress-mediated dopaminergic neuronal degeneration. Results: Expression of various Nox isoforms and cytoplasmic components were investigated in N27, rat dopaminergic cells. While most of Nox isoforms were constitutively expressed, Nox1 expression was significantly increased after treatment with 6-hydroxydopamine. Rac1, a key regulator in the Nox1 system, was also activated. Striatal injection of 6-hydroxydopamine increased Nox1 expression in dopaminergic neurons in the rat substantia nigra. Interestingly, it was localized into the nucleus, and immunostaining for DNA oxidative stress marker, 8-oxo-dG, was increased. Nox1expression was also found in the nucleus of dopaminergic neurons in the substantia nigra of Parkinson's disease patients. Adeno-associated virus-mediated Nox1 knockdown or Rac1 inhibition reduced 6-hydroxydopamine-induced oxidative DNA damage and dopaminergic neuronal degeneration significantly. Innovation: Nox1/Rac1 could serve as a potential therapeutic target for Parkinson's disease. Conclusion: We provide evidence that dopaminergic neurons are equipped with the Nox1/Rac1 superoxide-generating system. Stress-induced Nox1/Rac1 activation causes oxidative DNA damage and neurodegeneration. Reduced dopaminergic neuronal death achieved by targeting Nox1/Rac1, emphasizes the impact of oxidative stress caused by this system on the pathogenesis and therapy in Parkinson's disease. Antioxid. Redox Signal. 16, 1033–1045.
Blockade of angiotensin II type 1 (AT1) receptors ameliorates brain inflammation, and reduces excessive brain interleukin-1 beta (IL-1β) production and release from cortical microglia. The aim of this study was to determine whether, in addition, AT1 receptor blockade directly attenuates IL-1β-induced inflammatory responses in neuronal cultures.
SK-N-SH human neuroblasts and primary rat cortical neurons were pretreated with telmisartan followed by exposure to IL-1β. Gene expression was determined by reverse transcriptase (RT)-PCR, protein expression and kinase activation by western blotting, NADPH oxidase activity by the lucigenin method, prostaglandin E2 (PGE2) release by enzyme immunoassay, reactive oxygen species (ROS) generation by the dichlorodihydrofluorescein diacetate fluorescent probe assay, and peroxisome proliferator-activated receptor gamma (PPARγ) involvement was assessed with the antagonists GW9662 and T0070907, the agonist pioglitazone and the expression of PPARγ target genes ABCG1 and CD36.
We found that SK-N-SH neuroblasts expressed AT1 but not AT2 receptor mRNA. Telmisartan reduced IL-1β-induced cyclooxygenase-2 (COX-2) expression and PGE2 release more potently than did candesartan and losartan. Telmisartan reduced the IL-1β-induced increase in IL-1R1 receptor and NADPH oxidase-4 (NOX-4) mRNA expression, NADPH oxidase activity, and ROS generation, and reduced hydrogen peroxide-induced COX-2 gene expression. Telmisartan did not modify IL-1β-induced ERK1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation or nuclear factor-κB activation but significantly decreased IL-1β-induced c-Jun N-terminal kinase (JNK) and c-Jun activation. The JNK inhibitor SP600125 decreased IL-1β-induced PGE2 release with a potency similar to that of telmisartan. The PPARγ agonist pioglitazone reduced IL-1β-induced inflammatory reaction, whereas telmisartan did not activate PPARγ, as shown by its failure to enhance the expression of the PPARγ target genes ABCG1 and CD36, and the inability of the PPARγ antagonists GW9662 and T0070907 to modify the effect of telmisartan on COX-2 induction. The effect of telmisartan on IL-1β-stimulated COX-2 and IL-1R1 mRNA expression and ROS production was replicated in primary rat cortical neurons.
Telmisartan directly ameliorates IL-1β-induced neuronal inflammatory response by inhibition of oxidative stress and the JNK/c-Jun pathway. Our results support the hypothesis that AT1 receptor blockers are directly neuroprotective, and should be considered for the treatment of inflammatory conditions of the brain.
Angiotensin II AT1 receptor blockers; SK-N-SH neuroblasts; Cortical neurons; Neuronal inflammation; Neuroprotection; Oxidative stress; COX-2; PGE2 release; JNK activation; IL-1β neurotoxicity
NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date, seven members of this family have been reported, including Nox1-5 and Duox1 and -2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and it requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by small interfering RNA (siRNA) reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-guanine nucleotide exchange factor Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and abolish c-Src-induced Nox1 activity. Together, these results establish c-Src as an important regulator of Nox1 activity, and they may provide insight into the mechanisms of tumor formation in colon cancers.
NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5) gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs) are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT) and hypertensive subjects (HT). We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group). Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA), effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8) in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8%) than HT (−14.8±1.8). In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.
The schematic diagram shows that total cellular and apical membrane NOX5 proteins are greater than the other NOX proteins (NOX1, NOX2, and NOX4) in human renal proximal tubule (RPT) cells from hypertensive subjects. The increase in NOX5 expression is associated with increased reactive oxygen species production in these RPT cells. We suggest that the increased expression of NOX5, which is, in part, due to increased transcription, may be responsible for the increased oxidative stress in RPT cells in human essential hypertension.
•Basal levels of NOX5 mRNA and NOX5 protein in RPT cells were greater in HT than NT.•Basal level of NOX5 protein in apical membrane of RPT cells was greater in HT than NT.•NOX5-dependent oxidase activity, intra and extracellular ROS, and total membrane NADPH oxidase activity in RPT cells were greater in HT than NT.•D1-like receptor inhibition of NADPH oxidase activity is impaired in RPT cells from HT.
NOX5; ROS; Oxidative stress; Dopamine receptor
NADPH oxidase (NOX) is a multicomponent enzyme that mediates electron transfer from NADPH to molecular oxygen, which leads to the production of superoxide. NOX2/gp91phox is a catalytic subunit of NOX expressed in phagocytic cells. Several homologues of NOX2, including NOX1, have been identified in non-phagocytic cells. We investigated the contributory role of NOX1 and NOX2 in hepatic fibrosis. Hepatic fibrosis was induced in wild-type (WT) mice, NOX1-knockout (NOX1KO) mice, and NOX2-knockout (NOX2KO) mice by either CCl4 injections or bile duct ligation (BDL). The functional contribution of NOX1 and NOX2 in endogenous liver cells, including hepatic stellate cells (HSCs), and bone marrow (BM) derived cells, including Kupffer cells (KCs), to hepatic reactive oxygen species (ROS) generation and hepatic fibrosis was assessed in vitro and in vivo using NOX1- or NOX2-BM chimeric mice. Hepatic NOX1 and NOX2 mRNA expression was increased in the two experimental mouse models of hepatic fibrosis. While NOX1 was expressed in HSCs but not in KCs, NOX2 was expressed in both HSCs and KCs. Hepatic fibrosis and ROS generation were attenuated in both NOX1KO and NOX2KO mice after CCl4 or BDL. Liver fibrosis in chimeric mice indicated that NOX1 mediates the profibrogenic effects in endogenous liver cells, while NOX2 mediates the profibrogenic effects in both endogenous liver cells and BM-derived cells. Multiple NOX1 and NOX2 components were upregulated in activated HSCs. Both NOX1- and NOX2-deficient HSCs had decreased ROS generation and failed to upregulate collagen α1(I) and TGF-β in response to angiotensin II.
Both NOX1 and NOX2 in endogenous liver cells, including HSCs, have an important role in hepatic fibrosis, while NOX2 in BM-derived cells has a lesser role.
NADPH oxidase; oxidative stress; hepatic fibrosis; hepatic stellate cells
Calcium (Ca2+) handling proteins are known to play a pivotal role in the pathophysiology of cardiomyopathy. However little is known about early changes in the diabetic heart and the impact of insulin treatment (Ins).
Zucker Diabetic Fatty rats treated with or without insulin (ZDF ± Ins, n = 13) and lean littermates (controls, n = 7) were sacrificed at the age of 19 weeks. ZDF + Ins (n = 6) were treated with insulin for the last 6 weeks of life. Gene expression of Ca2+ ATPase in the cardiac sarcoplasmatic reticulum (SERCA2a, further abbreviated as SERCA) and phospholamban (PLB) were determined by northern blotting. Ca2+ transport of the sarcoplasmatic reticulum (SR) was assessed by oxalate-facilitated 45Ca-uptake in left ventricular homogenates. In addition, isolated neonatal cardiomyocytes were stimulated in cell culture with insulin, glucose or triiodthyronine (T3, positive control). mRNA expression of SERCA and PLB were measured by Taqman PCR. Furthermore, effects of insulin treatment on force of contraction and relaxation were evaluated by cardiomyocytes grown in a three-dimensional collagen matrix (engineered heart tissue, EHT) stimulated for 5 days by insulin. By western blot phosphorylations status of Akt was determed and the influence of wortmannin.
SERCA levels increased in both ZDF and ZDF + Ins compared to control (control 100 ± 6.2 vs. ZDF 152 ± 26.6* vs. ZDF + Ins 212 ± 18.5*# % of control, *p < 0.05 vs. control, #p < 0.05 vs. ZDF) whereas PLB was significantly decreased in ZDF and ZDF + Ins (control 100 ± 2.8 vs. ZDF 76.3 ± 13.5* vs. ZDF + Ins 79.4 ± 12.9* % of control, *p < 0.05 vs control). The increase in the SERCA/PLB ratio in ZDF and ZDF ± Ins was accompanied by enhanced Ca2+ uptake to the SR (control 1.58 ± 0.1 vs. ZDF 1.85 ± 0.06* vs. ZDF + Ins 2.03 ± 0.1* μg/mg/min, *p < 0.05 vs. control). Interestingly, there was a significant correlation between Ca2+ uptake and SERCA2a expression. As shown by in-vitro experiments, the effect of insulin on SERCA2a mRNA expression seemed to have a direct effect on cardiomyocytes. Furthermore, long-term treatment of engineered heart tissue with insulin increased the SERCA/PLB ratio and accelerated relaxation time. Akt was significantly phosphorylated by insulin. This effect could be abolished by wortmannin.
The current data demonstrate that early type 2 diabetes is associated with an increase in the SERCA/PLB ratio and that insulin directly stimulates SERCA expression and relaxation velocity. These results underline the important role of insulin and calcium handling proteins in the cardiac adaptation process of type 2 diabetes mellitus contributing to cardiac remodeling and show the important role of PI3-kinase-Akt-SERCA2a signaling cascade.
Diabetic heart; Insulin; SERCA expression; Relaxation velocity
Examine whether normalizing net hepatic glycogenesis restores endogenous glucose production and hepatic glucose phosphorylation in response to diabetic levels of plasma glucose and insulin in Zucker diabetic fatty rats (ZDF).
RESEARCH DESIGN AND METHODS
Hepatic glucose and intermediate fluxes (µmol ⋅ kg−1 ⋅ min−1) were measured with and without a glycogen phosphorylase inhibitor (GPI) using [2-3H]glucose, [3-3H]glucose, and [U-14C]alanine in 20 h-fasted conscious ZDF and their lean littermates (ZCL) under clamp conditions designed to maintain diabetic levels of plasma glucose and insulin.
With infusion of GPI into ZDF (ZDF-GPI+G), compared with vehicle infused ZDF (ZDF-V), high glycogen phosphorylase a activity was decreased and low synthase I activity was increased to that of ZCL. Low net glycogenesis from plasma glucose rose to 75% of ZCL levels (4 ± 1 in ZDF-V, 18 ± 1 in ZDF-GPI+G, and 24 ± 2 in ZCL) and phosphoenolpyruvate 260% (4 ± 2 in ZDF-V, 16 ± 1 in ZDF+GPI-G, and 6 ± 2 in ZCL). High endogenous glucose production was suppressed with GPI infusion but not to that of ZCL (46 ± 4 in ZDF-V, 18 ± 4 in ZDF-GPI+G, and −8 ± 3 in ZCL). This was accompanied by reduction of the higher glucose-6-phosphatase flux (75 ± 4 in ZDF-V, 41 ± 4 in ZDF-GPI+G, and 86 ± 12 in ZCL) and no change in low glucose phosphorylation or total gluconeogenesis.
In the presence of hyperglycemic-hyperinsulinemia in ZDF, reduced glycogenic flux partially contributes to a lack of suppression of hepatic glucose production by failing to redirect glucose-6-phosphate flux from production of glucose to glycogen but is not responsible for a lower rate of glucose phosphorylation.
In the majority of cell types, including the islet β-cell, transduction of extracellular signals involves ligand binding to a receptor, often followed by the activation G proteins and their effector modules. The islet β-cell is unusual in that glucose lacks an extracellular receptor. Instead, events consequent to glucose metabolism promote insulin secretion via the generation of diffusible second messengers and mobilization of calcium. A selective increase in intracellular calcium has been shown to regulate the phosphorylation status key islet proteins thereby facilitating insulin secretion. In addition to classical protein kinases [e.g., protein kinases A and C], recent studies from our laboratory have focused on the expression and function of various forms of NDPK/nm23-like histidine kinases in clonal β-cells, normal rodent, and human islets. Further, we recently reported localization of a cytosolic protein histidine phosphatase [PHP] in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knock down of nm23-H1 and PHP in insulin-secreting INS 832/13 cells significantly attenuated glucose-induced insulin secretion. We also observed significant alterations in the expression and function of nm23-H1/PHP in β-cells chronically exposed to elevated levels of glucose and saturated fatty acids, such as palmitate (i.e., glucolipotoxicity). Similar changes were also noted in islets from the Goto-Kakizaki and Zucker Diabetic Fatty rats, two known models for type 2 diabetes. It is concluded that protein histidine phosphorylation–dephosphorylation cycles play novel regulatory roles in G protein-mediated physiological insulin secretion and that abnormalities in this signaling axis lead to impaired insulin secretion in glucolipo-toxicity and type 2 diabetes.
Pancreatic islet; Insulin secretion; Histidine kinases; Histidine phosphatases; nm23
In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform–specific knockout mice. Both human and mouse β-cells express NOX, in particular NOX2. With use of Nox isoform–specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic β-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.
In the present study we investigated the interplay between matrix metalloproteinase 3 (MMP3) and NADPH oxidase 1 (Nox1) in the process of dopamine (DA) neuronal death. We found that MMP3 activation causes the induction of Nox1 via mitochondrial reactive oxygen species (ROS) production and subsequently Rac1 activation, eventually leading to Nox1-derived superoxide generation in a rat DA neuronal N27 cells exposed to 6-OHDA. While a MMP3 inhibitor, NNGH, largely attenuated mitochondrial ROS and subsequent Nox1 induction, both apocynin, a putative Nox inhibitor and GKT137831, a Nox1 selective inhibitor failed to reduce 6-OHDA-induced mitochondrial ROS. However, both inhibitors for MMP3 and Nox1 similarly attenuated 6-OHDA-induced N27 cell death. RNAi-mediated selective inhibition of MMP3 or Nox1 showed that knockdown of either MMP3 or Nox1 significantly reduced 6-OHDA-induced ROS generation in N27 cells. While 6-OHDA-induced Nox1 was abolished by MMP3 knockdown, Nox1 knockdown did not alter MMP3 expression. Direct overexpression of autoactivated MMP3 (actMMP3) in N27 cells or in rat substantia nigra (SN) increased expression of Nox1. Selective knockdown of Nox1 in the SN achieved by adeno-associated virus-mediated overexpression of Nox1-specific shRNA largely attenuated the actMMP3-mediated dopaminergic neuronal loss. Furthermore, Nox1 expression was significantly attenuated in Mmp3 null mice treated with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Together we established novel molecular mechanisms underlying oxidative stress-mediated dopaminergic neuronal death in which MMP3 activation is a key upstream event that leads to mitochondrial ROS, Nox1 induction and eventual dopaminergic neuronal death. Our findings may lead to the development of novel therapeutic approach.
The failure of pancreatic β cells to adapt to an increasing demand for insulin is the major mechanism by which patients progress from insulin resistance to type 2 diabetes (T2D) and is thought to be related to dysfunctional lipid homeostasis within those cells. In multiple animal models of diabetes, females demonstrate relative protection from β cell failure. We previously found that the hormone 17β-estradiol (E2) in part mediates this benefit. Here, we show that treating male Zucker diabetic fatty (ZDF) rats with E2 suppressed synthesis and accumulation of fatty acids and glycerolipids in islets and protected against β cell failure. The antilipogenic actions of E2 were recapitulated by pharmacological activation of estrogen receptor α (ERα) or ERβ in a rat β cell line and in cultured ZDF rat, mouse, and human islets. Pancreas-specific null deletion of ERα in mice (PERα–/–) prevented reduction of lipid synthesis by E2 via a direct action in islets, and PERα–/– mice were predisposed to islet lipid accumulation and β cell dysfunction in response to feeding with a high-fat diet. ER activation inhibited β cell lipid synthesis by suppressing the expression (and activity) of fatty acid synthase via a nonclassical pathway dependent on activated Stat3. Accordingly, pancreas-specific deletion of Stat3 in mice curtailed ER-mediated suppression of lipid synthesis. These data suggest that extranuclear ERs may be promising therapeutic targets to prevent β cell failure in T2D.
Diabetes mellitus is a major risk factor to impair endothelial function and induce cardiovascular diseases. TNF-alpha (TNF) is expressed during a variety of inflammatory conditions. We hypothesized that impairment in coronary endothelial function in type 2 diabetes is due to the overexpression of TNF and TNF receptors (TNFRs). Endothelium-dependent (acetylcholine, ACh) and –independent vasodilation (sodium nitroprusside, SNP) of isolated, pressurized (60 cmH2O) coronary arteries (50–100 μm) from lean control and Zucker diabetic fatty (ZDF, the model of type 2 diabetes) rats were determined. In lean rats, SNP and ACh induced dose-dependent vasodilation, but dilation to only ACh was blocked by the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 μM). In ZDF rats, dilation to ACh was blunted compared to lean rats, but SNP-induced dilation was comparable. Neutralizing antibodies to TNF, or blockade of NAD(P)H and xanthine oxidase, partially restored endothelium-dependent, NO-mediated vasodilation in isolated coronary arteries in ZDF rats, but anti-TNF did not alter endothelium-dependent vasodilation in lean rats. The mRNA expression of TNF receptor 1 (TNFR1, but not TNFR2) significantly increased in coronary arteries in ZDF rats. Protein expression of TNF and N-Tyr (ONOO−) were higher in coronary arteries in ZDF than those in lean rats. Production of H2O2, NAD(P)H oxidase and xanthine oxidase activity were all higher in ZDF rats than those in lean controls; anti-TNF reduces the production of H2O2, N-Tyr expression, NAD(P)H oxidase and xanthine oxidase activity in ZDF rats. These results demonstrate the endothelial dysfunction occurring in type 2 diabetes is the result of effects of the inflammatory cytokine TNF that activates NAD(P)H oxidase and xanthine oxidase; and perhaps acts mainly through the overexpression of TNFR1.
Microcirculation; coronary artery disease; nitric oxide
Obesity-associated inflammation causes insulin resistance. Obese adipose tissue displays hypertrophied adipocytes and increased expression of the cannabinoid-1 receptor. Cobalt protoporphyrin (CoPP) increases heme oxygenase-1 (HO-1) activity, increasing adiponectin and reducing inflammatory cytokines. We hypothesize that CoPP administration to Zucker diabetic fat (ZDF) rats would improve insulin sensitivity and remodel adipose tissue. Twelve-week-old Zucker lean and ZDF rats were divided into 4 groups: Zucker lean, Zucker lean–CoPP, ZDF, and ZDF–CoPP. Control groups received vehicle and treatment groups received CoPP (2 mg/kg body weight) once weekly for 6 weeks. Serum insulin levels and glucose response to insulin injection were measured. At 18 weeks of age, rats were euthanized, and aorta, kidney, and subcutaneous and visceral adipose tissues were harvested. HO-1 expression was measured by Western blot analysis and HO-1 activity by serum carbon monoxide content. Adipocyte size and cannabinoid-1 expression were measured. Adipose tissue volumes were determined using MRI. CoPP significantly increased HO-1 activity, phosphorylated AKT and phosphorylated AMP kinase, and serum adiponectin in ZDF rats. HO-1 induction improved hyperinsulinemia and insulin sensitivity in ZDF rats. Subcutaneous and visceral adipose tissue volumes were significantly decreased in ZDF rats. Adipocyte size and cannabinoid-1 expression were both significantly reduced in ZDF–CoPP rats in subcutaneous and visceral adipose tissues. This study demonstrates that HO-1 induction improves insulin sensitivity, downregulates the peripheral endocannabinoid system, reduces adipose tissue volume, and causes adipose tissue remodeling in a model of obesity-induced insulin resistance. These findings suggest HO-1 as a potential therapeutic target for obesity and its associated health risks.
insulin resistance; heme oxygenase-1; adiponectin; adiposity; endocannabinoid; pAMPK
NADPH oxidase (Nox)–dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D1-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58±3.3%) than in hypertensive rats (31±5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67phox in cells from normotensive rats. D1-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl–β-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats.
NADPH oxidase; dopamine receptor; reactive oxygen species; lipid rafts
Phagocyte-like NADPH oxidase (Nox2) has been shown to play regulatory roles in the metabolic dysfunction of the islet β-cell under the duress of glucolipotoxic conditions and exposure to proinflammatory cytokines. However, the precise mechanisms underlying Nox2 activation by these stimuli remain less understood. To this end, we report a time-dependent phosphorylation of p47phox, a cytosolic subunit of Nox2, by cytomix (IL-1β+TNFα+IFNγ) in insulin-secreting INS-1 832/13 cells. Furthermore, cytomix induced the expression of gp91phox, a membrane component of Nox2. 2-Bromopalmitate (2-BP), a known inhibitor of protein palmitoylation, markedly attenuated cytokine-induced, Nox2-mediated reactive oxygen species (ROS) generation and inducible nitric oxide synthase-mediated nitric oxide (NO) generation. However, 2-BP failed to exert any significant effects on cytomix-induced CHOP expression, a marker for endoplasmic reticulum stress. Together, our findings identify palmitoyltransferase as a target for inhibition of cytomix-induced oxidative (ROS generation) and nitrosative (NO generation) stress in the pancreatic β-cell.
Pancreatic β-cell; NADPH-oxidase; 2-Bromopalmitate; Cytokines; Oxidative stress; Nitrosative stress
The reactive oxygen-generating NADPH oxidases (Noxes) function in a variety of biological roles, and can be broadly classified into those that are regulated by subunit interactions and those that are regulated by calcium. The prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the integral membrane protein, p22phox, and this heterodimer binds to the regulatory subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1 (NOXA1), respective homologs of p47phox and p67phox, together with p22phox and Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary history of these subunits.
Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, the unicellular organism that is the closest relatives of multicellular animals, encodes early prototypes of p22phox, p47phox as well as the earliest known Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. Duplication of primordial p47phox and p67phox genes occurred in vertebrates, with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of characteristic domains of regulatory subunits suggests a novel view of the evolution of Nox: in fish, p40phox participated in regulating both Nox1 and Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an autoinhibitory region that is characteristic of mammalian p47phox, and this was subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence comparisons identified both putative key residues conserved in characteristic domains and previously unidentified conserved regions. Also, candidate organizer/activator proteins in fungi and amoeba are identified and hypothetical activation models are suggested.
This is the first report to provide the comprehensive view of the molecular evolution of regulatory subunits for Nox enzymes. This approach provides clues for understanding the evolution of biochemical and physiological functions for regulatory-subunit-dependent Nox enzymes.