Smooth Muscle Nitric Oxide Responsiveness and Clinical Maturation of Hemodialysis Arteriovenous Fistulae.

The arteriovenous fistula is the preferred type of hemodialysis vascular access for patients with end-stage renal disease, but a high proportion of newly created fistulas fail to mature for use. Stenosis caused by neointimal hyperplasia often is present in fistulas with maturation failure, suggesting that local mechanisms controlling vascular smooth muscle cell (SMC) migration and proliferation are important contributors to maturation failure. SMCs cultured from explants of vein tissue obtained at the time of fistula creation from 19 patients with end-stage renal disease were studied to determine whether smooth muscle responsiveness to nitric oxide is associated with fistula maturation outcomes. Nitric oxide-induced inhibition of smooth muscle cell migration, but not proliferation, was greater in cells from patients with subsequent fistula maturation success than from patients with subsequent fistula maturation failure (mean inhibition percentage, 17 versus 5.7, respectively; P = 0.035). Impaired nitric oxide responsiveness was associated with oxidation of the calcium regulatory protein, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and was reversed by overexpressing SERCA (1.8-fold increase in inhibition, P = 0.0128) or down-regulating Nox4-based NADPH oxidase (2.3-fold increase in inhibition; P = 0.005). Our data suggest that the nitric oxide responsiveness of SMC migration is associated with fistula maturation success and raises the possibility that therapeutic restoration of nitric oxide responsiveness through manipulation of local mediators may prevent fistula maturation failure.

Paracrine Wnt1 Drives Interstitial Fibrosis without Inflammation by Tubulointerstitial Cross-Talk.

AKI with incomplete epithelial repair is a major contributor to CKD characterized by tubulointerstitial fibrosis. Injury-induced epithelial secretion of profibrotic factors is hypothesized to underlie this link, but the identity of these factors and whether epithelial injury is required remain undefined. We previously showed that activation of the canonical Wnt signaling pathway in interstitial pericytes cell autonomously drives myofibroblast activation in vivo. Here, we show that inhibition of canonical Wnt signaling also substantially prevented TGFß-dependent myofibroblast activation in vitro. To investigate whether Wnt ligand derived from proximal tubule is sufficient for renal fibrogenesis, we generated a novel mouse strain with inducible proximal tubule Wnt1 secretion. Adult mice were treated with vehicle or tamoxifen and euthanized at 12 or 24 weeks postinjection. Compared with vehicle-treated controls, kidneys with tamoxifen-induced Wnt1 expression from proximal tubules displayed interstitial myofibroblast activation and proliferation and increased matrix protein production. PDGF receptor ß-positive myofibroblasts isolated from these kidneys exhibited increased canonical Wnt target gene expression compared with controls. Notably, fibrotic kidneys had no evidence of inflammatory cytokine expression, leukocyte infiltration, or epithelial injury, despite the close histologic correlation of each with CKD. These results provide the first example of noninflammatory renal fibrosis. The fact that epithelial-derived Wnt ligand is sufficient to drive interstitial fibrosis provides strong support for the maladaptive repair hypothesis in the AKI to CKD transition.

A redox-resistant sirtuin-1 mutant protects against hepatic metabolic and oxidant stress.

Sirtuin-1 (SirT1), a member of the NAD(+)-dependent class III histone deacetylase family, is inactivated in vitro by oxidation of critical cysteine thiols. In a model of metabolic syndrome, SirT1 activation attenuated apoptosis of hepatocytes and improved liver function including lipid metabolism. We show in SirT1-overexpressing HepG2 cells that oxidants (nitrosocysteine and hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivated SirT1 by reversible oxidative post-translational modifications (OPTMs) on three cysteines. Mutating these oxidation-sensitive cysteines to serine preserved SirT1 activity and abolished reversible OPTMs. Overexpressed mutant SirT1 maintained deacetylase activity and attenuated proapoptotic signaling, whereas overexpressed wild type SirT1 was less protective in metabolically or oxidant-stressed cells. To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 was overexpressed to remove this modification. Glutaredoxin-1 overexpression maintained endogenous SirT1 activity and prevented proapoptotic signaling in metabolically stressed HepG2 cells. The in vivo significance of oxidative inactivation of SirT1 was investigated in livers of high fat diet-fed C57/B6J mice. SirT1 deacetylase activity was decreased in the absence of changes in SirT1 expression and associated with a marked increase in OPTMs. These results indicate that glutathione adducts on specific SirT1 thiols may be responsible for dysfunctional SirT1 associated with liver disease in metabolic syndrome.

Nox2 mediates high fat high sucrose diet-induced nitric oxide dysfunction and inflammation in aortic smooth muscle cells.

Diet-induced obesity and metabolic syndrome are important contributors to cardiovascular diseases. The decreased nitric oxide (NO) bioactivity in endothelium and the impaired response of smooth muscle cell (SMC) to NO significantly contribute to vascular pathologies, including atherosclerosis and arterial restenosis after angioplasty. Sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is an important mediator of NO function in both endothelial cells and SMCs, and its irreversible oxidation impairs its stimulation by NO. We used C57BL/6J mice fed a high fat high sucrose diet (HFHSD) to study the role of SMC SERCA in diet-induced obesity and metabolic syndrome. We found that HFHSD upregulated Nox2 based NADPH oxidase, induced inflammation, increased irreversible SERCA oxidation, and suppressed the response of aortic SERCA to NO. Cultured aortic SMCs from mice fed HFHSD showed increased reactive oxygen species production, Nox2 upregulation, irreversible SERCA oxidation, inflammation, and a decreased ability of NO to inhibit SMC migration. Overexpression of wild type SERCA2b or downregulation of Nox2 restored NO-mediated inhibition of migration in SMCs isolated from HFHSD-fed mice. In addition, tumor necrosis factor alpha (TNFα) increased Nox2 which induced SERCA oxidation and inflammation. Taken together, Nox2 induced by HFHSD plays significant roles in controlling SMC responses to NO and TNFα-mediated inflammation, which may contribute to the development of cardiovascular diseases in diet-induced obesity and metabolic syndrome.

Endomembrane H-Ras controls vascular endothelial growth factor-induced nitric-oxide synthase-mediated endothelial cell migration.

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling.

The polyphenols resveratrol and S17834 prevent the structural and functional sequelae of diet-induced metabolic heart disease in mice.

BACKGROUND: Diet-induced obesity is associated with metabolic heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. Polyphenols such as resveratrol and the synthetic flavonoid derivative S17834 exert beneficial systemic and cardiovascular effects in a variety of settings including diabetes mellitus and chronic hemodynamic overload. METHODS AND RESULTS: We characterized the structural and functional features of a mouse model of diet-induced metabolic syndrome and used the model to test the hypothesis that the polyphenols prevent myocardial hypertrophy and diastolic dysfunction. Male C57BL/6J mice were fed a normal diet or a diet high in fat and sugar (HFHS) with or without concomitant treatment with S17834 or resveratrol for up to 8 months. HFHS diet-fed mice developed progressive left ventricular hypertrophy and diastolic dysfunction with preservation of systolic function in association with myocyte hypertrophy and interstitial fibrosis. In HFHS diet-fed mice, there was increased myocardial oxidative stress with evidence of oxidant-mediated protein modification via tyrosine nitration and 4-OH-2-nonenol adduction. HFHS diet-fed mice also exhibited increases in plasma fasting glucose, insulin, and homeostasis model assessment of insulin resistance indicative of insulin resistance. Treatment with S17834 or resveratrol prevented left ventricular hypertrophy and diastolic dysfunction. For S17834, these beneficial effects were associated with decreases in oxidant-mediated protein modifications and hyperinsulinemia and increased plasma adiponectin. CONCLUSIONS: Resveratrol and S17834 administered concurrently with a HFHS diet prevent the development of left ventricular hypertrophy, interstitial fibrosis, and diastolic dysfunction. Multiple mechanisms may contribute to the beneficial effects of the polyphenols, including a reduction in myocardial oxidative stress and related protein modifications, amelioration of insulin resistance, and increased plasma adiponectin. The polyphenols resveratrol and S17834 may be of value in the prevention of diet-induced metabolic heart disease.

SERCA Cys674 sulphonylation and inhibition of L-type Ca2+ influx contribute to cardiac dysfunction in endotoxemic mice, independent of cGMP synthesis.

The goal of this study was to identify the cellular mechanisms responsible for cardiac dysfunction in endotoxemic mice. We aimed to differentiate the roles of cGMP [produced by soluble guanylyl cyclase (sGC)] versus oxidative posttranslational modifications of Ca(2+) transporters. C57BL/6 mice [wild-type (WT) mice] were administered lipopolysaccharide (LPS; 25 µg/g ip) and euthanized 12 h later. Cardiomyocyte sarcomere shortening and Ca(2+) transients (ΔCai) were depressed in LPS-challenged mice versus baseline. The time constant of Ca(2+) decay (τCa) was prolonged, and sarcoplasmic reticulum Ca(2+) load (CaSR) was depressed in LPS-challenged mice (vs. baseline), indicating decreased activity of sarco(endo)plasmic Ca(2+)-ATPase (SERCA). L-type Ca(2+) channel current (ICa,L) was also decreased after LPS challenge, whereas Na(+)/Ca(2+) exchange activity, ryanodine receptors leak flux, or myofilament sensitivity for Ca(2+) were unchanged. All Ca(2+)-handling abnormalities induced by LPS (the decrease in sarcomere shortening, ΔCai, CaSR, ICa,L, and τCa prolongation) were more pronounced in mice deficient in the sGC main isoform (sGCα1(-/-) mice) versus WT mice. LPS did not alter the protein expression of SERCA and phospholamban in either genotype. After LPS, phospholamban phosphorylation at Ser(16) and Thr(17) was unchanged in WT mice and was increased in sGCα1(-/-) mice. LPS caused sulphonylation of SERCA Cys(674) (as measured immunohistochemically and supported by iodoacetamide labeling), which was greater in sGCα1(-/-) versus WT mice. Taken together, these results suggest that cardiac Ca(2+) dysregulation in endotoxemic mice is mediated by a decrease in L-type Ca(2+) channel function and oxidative posttranslational modifications of SERCA Cys(674), with the latter (at least) being opposed by sGC-released cGMP.

Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver.

Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of human type 2 diabetes (T2DM). Although SIRT1 has a therapeutic effect on metabolic deterioration in T2DM, the precise mechanisms by which SIRT1 improves insulin resistance remain unclear. Here, we demonstrate that adenovirus-mediated overexpression of SIRT1 in the liver of diet-induced insulin-resistant low-density lipoprotein receptor-deficient mice and of genetically obese ob/ob mice attenuates hepatic steatosis and ameliorates systemic insulin resistance. These beneficial effects were associated with decreased mammalian target of rapamycin complex 1 (mTORC1) activity, inhibited the unfolded protein response (UPR), and enhanced insulin receptor signaling in the liver, leading to decreased hepatic gluconeogenesis and improved glucose tolerance. The tunicamycin-induced splicing of X-box binding protein-1 and expression of GRP78 and CHOP were reduced by resveratrol in cultured cells in a SIRT1-dependent manner. Conversely, SIRT1-deficient mouse embryonic fibroblasts challenged with tunicamycin exhibited markedly increased mTORC1 activity and impaired ER homeostasi and insulin signaling. These effects were abolished by mTORC1 inhibition by rapamycin in human HepG2 cells. These studies indicate that SIRT1 serves as a negative regulator of UPR signaling in T2DM and that SIRT1 attenuates hepatic steatosis, ameliorates insulin resistance, and restores glucose homeostasis, largely through the inhibition of mTORC1 and ER stress.

Upregulation of Nox4 by TGF{beta}1 oxidizes SERCA and inhibits NO in arterial smooth muscle of the prediabetic Zucker rat.

RATIONALE: Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by diabetes mellitus. OBJECTIVE: To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied. METHODS AND RESULTS: In culture, ZO aortic SMCs showed a significant increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL). The sarco-/endoplasmic reticulum Ca(2+) ATPase (SERCA) nitrotyrosine-294,295 and cysteine-674 (C674)-SO(3)H were increased in ZO SMCs, indicating oxidant stress. Unlike ZL SMC, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO. Transfection of Nox4 small interference RNA or overexpression of SERCA2b wild type, but not C674S mutant SERCA, restored the response to NO. Knockdown of Nox4 also decreased SERCA oxidation in ZO SMCs. In addition, transforming growth factor-ß1 via Smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the antimigratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO(3)H were significantly increased in ZO aorta. After common carotid artery injury, knockdown of Nox4 by adenoviral Nox4 short hairpin RNA decreased Nox4 and SERCA C674-SO(3)H staining and significantly decreased injury-induced neointima. CONCLUSION: These studies indicate that the upregulation of Nox4 by transforming growth factor-ß1 in ZO SMCs is responsible for the impaired response to NO by a mechanism involving the oxidation of SERCA C674. Knockdown of Nox4 inhibits oxidation of SERCA, as well as neointima formation, after ZO common carotid artery injury.

Angiotensin II modulates interleukin-1ß-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-κB crosstalk.

Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1ß-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-κB, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1ß-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1ß, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE(-/-)) mice. VCAM-1 and iNOS expression were higher in ApoE(-/-) than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE(-/-) mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells through influencing ERK-NF-κB crosstalk, which may contribute to angiotensin II-induced inflammatory disorders related to cardiovascular diseases.

High-fat diet increases and the polyphenol, S17834, decreases acetylation of the sirtuin-1-dependent lysine-382 on p53 and apoptotic signaling in atherosclerotic lesion-prone aortic endothelium of normal mice.

Our purpose was to determine if high-fat diet and treatment with a polyphenol regulate the acetylation of lysine-382 of p53, the site regulated by sirtuin-1, and apoptosis in the endothelium of the atherosclerotic lesion-prone mouse aortic arch. In cultured endothelial cells, 2 atherogenic stimuli, hydrogen peroxide and tumor necrosis factor-α, increased the acetylation of p53 lysine-382, and caspase-3 cleavage, an indicator of apoptotic signaling. The polyphenol, S17834, significantly prevented these changes. In low-density lipoprotein receptor-deficient mice, a high-fat diet increased, and treatment with S17834 attenuated early atherosclerotic lesions on the lesser curvature of the aortic arch. In wild-type C57BL6 mice fed the same diet, no atherosclerotic lesions were observed in this lesion-prone area, but p53 acetylation and caspase-3 cleavage increased in the endothelium. In high-fat fed mice, S17834 increased sirtuin-1 protein in the lesion-prone endothelium and prevented both the increase in p53 acetylation and caspase-3 cleavage without affecting blood lipids. These results indicate that high-fat diet increases and S17834 decreases the acetylation of p53 in lesion-prone aortic endothelial cells of normal mice independently of blood lipids, suggesting that the polyphenol may regulate endothelial cell p53 acetylation and apoptosis via local actions.

Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-deficient mice.

Because polyphenols may have beneficial effects on dyslipidemia, which accelerates atherosclerosis in diabetes, we examined the effect of polyphenols on hepatocellular AMP-activated protein kinase (AMPK) activity and lipid levels, as well as hyperlipidemia and atherogenesis in type 1 diabetic LDL receptor-deficient mice (DMLDLR(-/-)). In HepG2 hepatocytes, polyphenols, including resveratrol (a major polyphenol in red wine), apigenin, and S17834 (a synthetic polyphenol), increased phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC), and they increased activity of AMPK with 200 times the potency of metformin. The polyphenols also prevented the lipid accumulation that occurred in HepG2 cells exposed to high glucose, and their ability to do so was mimicked and abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Furthermore, treatment of DMLDLR(-/-) mice with S17834 prevented the decrease in AMPK and ACC phosphorylation and the lipid accumulation in the liver, and it also inhibited hyperlipidemia and the acceleration of aortic lesion development. These studies 1) reveal that inactivation of hepatic AMPK is a key event in the pathogenesis of hyperlipidemia in diabetes, 2) point to a novel mechanism of action of polyphenols to lower lipids by activating AMPK, and 3) emphasize a new therapeutic avenue to benefit hyperlipidemia and atherosclerosis specifically in diabetes via activating AMPK.

S-glutathiolation of p21ras by peroxynitrite mediates endothelial insulin resistance caused by oxidized low-density lipoprotein.

OBJECTIVE: To understand the mechanism by which oxidants are linked to insulin resistance, bovine aortic endothelial cells were exposed to oxidized low-density lipoproteins (oxLDL) or peroxynitrite. METHODS AND RESULTS: OxLDL transiently increased phosphorylation of Erk and Akt within 5 minutes, but 60 minutes later, resulted in decreased insulin-induced Akt phosphorylation. OxLDL promoted a 2- to 5-fold increase in oxidant generation as measured by dihydrorhodamine or dihydroethidium oxidation that was ascribed to peroxynitrite. Exogenous peroxynitrite (25 to 100 micromol/L) or oxidized glutathione mimicked the effects of oxLDL. OxLDL increased the S-glutathiolation of p21ras, and adenoviral transfection with either a mutant p21ras (C118S) lacking the predominant site of S-glutathiolation or a dominant-negative mutant restored insulin-induced Akt phosphorylation. The requirement for oxidant-mediated S-glutathiolation and activation of p21ras in mediating insulin resistance was further implicated by showing that insulin signaling was restored by Mek inhibitors or by overexpression of glutaredoxin-1. Furthermore, oxLDL increased Erk-dependent phosphorylation of insulin receptor substrate-1 serine-616 that was prevented by inhibiting oxidant generation, Erk activation, or by the p21ras C118S mutant. CONCLUSIONS: This study provides direct evidence for a novel molecular mechanism by which oxidants can induce insulin resistance via S-glutathiolation of p21ras and Erk-dependent inhibition of insulin signaling.

Glutaredoxin-1 Deficiency Causes Fatty Liver and Dyslipidemia by Inhibiting Sirtuin-1.

AIMS: Nonalcoholic fatty liver (NAFL) is a common liver disease associated with metabolic syndrome, obesity, and diabetes that is rising in prevalence worldwide. Various molecular perturbations of key regulators and enzymes in hepatic lipid metabolism cause NAFL. However, redox regulation through glutathione (GSH) adducts in NAFL remains largely elusive. Glutaredoxin-1 (Glrx) is a small thioltransferase that removes protein GSH adducts without having direct antioxidant properties. The liver contains abundant Glrx but its metabolic function is unknown. RESULTS: Here we report that normal diet-fed Glrx-deficient mice (Glrx-/-) spontaneously develop obesity, hyperlipidemia, and hepatic steatosis by 8 months of age. Adenoviral Glrx repletion in the liver of Glrx-/- mice corrected lipid metabolism. Glrx-/- mice exhibited decreased sirtuin-1 (SirT1) activity that leads to hyperacetylation and activation of SREBP-1 and upregulation of key hepatic enzymes involved in lipid synthesis. We found that GSH adducts inhibited SirT1 activity in Glrx-/- mice. Hepatic expression of nonoxidizable cysteine mutant SirT1 corrected hepatic lipids in Glrx-/- mice. Wild-type mice fed high-fat diet develop metabolic syndrome, diabetes, and NAFL within several months. Glrx deficiency accelerated high-fat-induced NAFL and progression to steatohepatitis, manifested by hepatic damage and inflammation. INNOVATION: These data suggest an essential role of hepatic Glrx in regulating SirT1, which controls protein glutathione adducts in the pathogenesis of hepatic steatosis. CONCLUSION: We provide a novel redox-dependent mechanism for regulation of hepatic lipid metabolism, and propose that upregulation of hepatic Glrx may be a beneficial strategy for NAFL. Antioxid. Redox Signal. 27, 313-327.

IκBß attenuates angiotensin II-induced cardiovascular inflammation and fibrosis in mice.

The development of cardiovascular fibrosis is associated with chronic inflammation, where activation of nuclear factor κB (NF-κB) signaling may play a critical role. NF-κB activation is tightly regulated by the cellular inhibitor of κB (IκB) family of proteins, such as IκBα and IκBß. IκBα and IκBß display different regulation kinetics in response to inflammatory stimulation. The present study tested the hypothesis that IκBα and IκBß may have different roles in modulating cardiovascular inflammation and fibrosis, using a model of angiotensin II infusion-induced hypertension in wild-type mice and IκBß knock-in mice, in which the IκBα gene is replaced by IκBß cDNA (AKBI). In WT mice, subcutaneous angiotensin II infusion for 7 days induced increased perivascular and interstitial collagen deposition and fibrotic lesions, associated with myocardial interstitial hemosiderin accumulation and extensive macrophage infiltration. These effects of angiotensin II were dramatically limited in AKBI mice. Replacement of IκBα with IκBß significantly attenuated angiotensin II infusion-induced expression of interleukin 1ß, interleukin 6, monocyte chemotactic protein 1, collagen I and III, fibronectin, and tissue inhibitor of metalloproteinase 1 in the hearts. Furthermore, using cultured vascular smooth muscle cells, we demonstrated that interleukin 1ß-induced NF-κB activation and monocyte chemotactic protein 1, vascular cell adhesion molecule 1, and tissue inhibitor of metalloproteinase 1 expressions were suppressed in the AKBI cells because of the replacement of IκBα with IκBß. These results indicate that NF-κB has an essential role in mediating the cardiovascular inflammatory response to angiotensin II and suggest that targeting the balance of IκBα and IκBß expression might be a novel therapeutic modality in preventing fibrosis in hypertensive cardiovascular disease.

AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.

AMPK has emerged as a critical mechanism for salutary effects of polyphenols on lipid metabolic disorders in type 1 and type 2 diabetes. Here we demonstrate that AMPK interacts with and directly phosphorylates sterol regulatory element binding proteins (SREBP-1c and -2). Ser372 phosphorylation of SREBP-1c by AMPK is necessary for inhibition of proteolytic processing and transcriptional activity of SREBP-1c in response to polyphenols and metformin. AMPK stimulates Ser372 phosphorylation, suppresses SREBP-1c cleavage and nuclear translocation, and represses SREBP-1c target gene expression in hepatocytes exposed to high glucose, leading to reduced lipogenesis and lipid accumulation. Hepatic activation of AMPK by the synthetic polyphenol S17834 protects against hepatic steatosis, hyperlipidemia, and accelerated atherosclerosis in diet-induced insulin-resistant LDL receptor-deficient mice in part through phosphorylation of SREBP-1c Ser372 and suppression of SREBP-1c- and -2-dependent lipogenesis. AMPK-dependent phosphorylation of SREBP may offer therapeutic strategies to combat insulin resistance, dyslipidemia, and atherosclerosis.

Redox regulation of sirtuin-1 by S-glutathiolation.

Sirtuin-1 (SIRT1) is an NAD(+)-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepharose, and mass spectrometry identified several cysteines that are modified by GSNO, including Cys-67 which was S-glutathiolated. GSNO had no effect on basal SIRT1 deacetylase activity, but inhibited stimulation of activity by resveratrol (RSV) with an IC(50) of 69 microM. These observations indicate that S-glutathiolation of SIRT1 by low concentrations of reactive glutathione can modulate its enzymatic activity.

SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase.

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser(428), and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKbeta, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-kappaB through phosphorylation of IkappaBbeta.

Activation of NF-kappaB requires the phosphorylation and degradation of its associated inhibitory proteins, IkappaB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1beta to induce persistent activation of NF-kappaB in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-kappaB activation. In cultured rat VSMCs, IL-1beta activated ERK and induced degradation of both IkappaBalpha and IkappaBbeta, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-kappaB p65. RSK1, a downstream kinase of ERK, was associated with an IkappaBbeta/NF-kappaB complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1beta decreased IkappaBbeta in the RSK1/IkappaBbeta/NF-kappaB complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1beta-induced IkappaBbeta decrease without influencing ether ERK phosphorylation or the earlier IkappaBalpha degradation. By using recombinant wild-type and mutant IkappaBbeta proteins, both active ERK2 and RSK1 were found to directly phosphorylate IkappaBbeta, but only active RSK1 phosphorylated IkappaBbeta on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IkappaBbeta and contributes to the persistent activation of NF-kappaB by IL-1beta.