Apigenin targets fetuin-A to ameliorate obesity-induced insulin resistance.

Fetuin-A, a hepatokine secreted by hepatocytes, binds to insulin receptors and consequently impairs the activation of the insulin signaling pathway, leading to insulin resistance. Apigenin, a flavonoid isolated from plants, has beneficial effects on insulin resistance; however, its regulatory mechanisms are not fully understood. In the present study, we investigated the molecular mechanisms underlying the protective effects of apigenin on insulin resistance. In Huh7 cells, treatment with apigenin decreased the mRNA expression of fetuin-A by decreasing reactive oxygen species-mediated casein kinase 2α (CK2α)-nuclear factor kappa-light-chain-enhancer of activated B activation; besides, apigenin decreased the levels of CK2α-dependent fetuin-A phosphorylation and thus promoted fetuin-A degradation through the autophagic pathway, resulting in a decrease in the protein levels of fetuin-A. Moreover, apigenin prevented the formation of the fetuin-A-insulin receptor (IR) complex and thereby rescued the PA-induced impairment of the insulin signaling pathway, as evidenced by increased phosphorylation of IR substrate-1 and Akt, and translocation of glucose transporter 2 from the cytosol to the plasma membrane. Similar results were observed in the liver of HFD-fed mice treated with apigenin. Collectively, our findings revealed that apigenin ameliorates obesity-induced insulin resistance in the liver by targeting fetuin-A.

Di-(2-ethylhexyl) Phthalate Limits the Lipid-Lowering Effects of Simvastatin by Promoting Protein Degradation of Low-Density Lipoprotein Receptor: Role of PPARγ-PCSK9 and LXRα-IDOL Signaling Pathways.

Dialysis prevents death from uremia in patients with end-stage renal disease (ESRD). Nevertheless, during hemodialysis, circulating levels of di-(2-ethylhexyl) phthalate (DEHP) are increased due to phthalates leaching from medical tubes. Statins are an effective therapy for reducing the risks associated with cardiovascular diseases in patients with chronic kidney disease; however, the mechanism by which statins fail to reduce cardiovascular events in hemodialysis ESRD patients remains unclear. In this study, we investigated whether DEHP and its metabolites interfere with the lipid-lowering effect of statins in hepatocytes. In Huh7 cells, treatment with DEHP and its metabolites abolished the simvastatin-conferred lipid-lowering effect. Mechanistically, DEHP down-regulated the expression of low-density lipoprotein receptor (LDLR) and led to a decrease in LDL binding, which was mediated by the activation of the PPARγ-PCSK9 and LXRα-IDOL signaling pathways. Additionally, the NOX-ROS-TRPA1 pathway is involved in the DEHP-mediated inhibition of LDLR expression and LDL binding activity. Blockage of this pathway abrogated the DEHP-mediated inhibition in the LDLR expression and LDL binding of simvastatin. Collectively, DEHP induces the activation of the NOX-ROS-TRPA1 pathway, which in turn activates PPARγ-PCSK9- and LXRα-IDOL-dependent signaling, and, ultimately, diminishes the statin-mediated lipid-lowering effect in hepatocytes.

Ractopamine at legal residue dosage accelerates atherosclerosis by inducing endothelial dysfunction and promoting macrophage foam cell formation.

Ractopamine, a synthetic ß-adrenoreceptor agonist, is used as an animal feed additive to increase food conversion efficiency and accelerate lean mass accretion in farmed animals. The U.S. Food and Drug Administration claimed that ingesting products containing ractopamine residues at legal dosages might not cause short-term harm to human health. However, the effect of ractopamine on chronic inflammatory diseases and atherosclerosis is unclear. Therefore, we investigated the effects of ractopamine on atherosclerosis and its action mechanism in apolipoprotein E-null (apoe-/-) mice and human endothelial cells (ECs) and macrophages. Daily treatment with ractopamine for four weeks increased the body weight and the weight of brown adipose tissues and gastrocnemius muscles. However, it decreased the weight of white adipose tissues in apoe-/- mice. Additionally, ractopamine exacerbated hyperlipidemia and systemic inflammation, deregulated aortic cholesterol metabolism and inflammation, and accelerated atherosclerosis. In ECs, ractopamine treatment induced endothelial dysfunction and increased monocyte adhesion and transmigration across ECs. In macrophages, ractopamine dysregulated cholesterol metabolism by increasing oxidized low-density lipoprotein (oxLDL) internalization and decreasing reverse cholesterol transporters, increasing oxLDL-induced lipid accumulation. Collectively, our findings revealed that ractopamine induces EC dysfunction and deregulated cholesterol metabolism of macrophages, which ultimately accelerates atherosclerosis progression.

New Mechanisms of Bromelain in Alleviating Non-Alcoholic Fatty Liver Disease-Induced Deregulation of Blood Coagulation.

Bromelain, an enzyme extracted from the stems of pineapples, exerts anticoagulant effects; however, the regulatory mechanisms are not fully understood. Here, we aimed to investigate the effects of bromelain on non-alcoholic fatty liver disease (NAFLD)-induced deregulation of blood coagulation and the underlying molecular mechanisms. C57BL/6 mice were fed a high-fat diet (HFD), with or without bromelain (20 mg/kg/day) administration, for 12 weeks. Treatment with bromelain decreased thrombus formation in the liver and prolonged HFD-induced shortened prothrombin, activated partial thromboplastin, and fibrinogen times. Moreover, liquid chromatography-mass spectrometry/mass spectrometry and Western blot analysis showed that bromelain inhibited NAFLD-induced activation of the intrinsic, extrinsic, and common pathways by upregulating the protein expression of antithrombin III, serpin family G member 1, and α1-antitrypsin, and downregulating the protein expression of fibrinogen in the liver and plasma. Bromelain also upregulated the level of plasminogen and downregulating factor XIII expression in the liver and plasma. Collectively, these findings suggest that bromelain exerts anticoagulant effects on NAFLD-induced deregulation of coagulation by inhibiting the activation of the coagulation cascade, decreasing the stability of clots, and promoting fibrinolytic activity. The present study provides new insights into the potential therapeutic value of bromelain for the prevention and treatment of thrombosis-related diseases.

Genetic Deletion of HLJ1 Does Not Affect Blood Coagulation in Mice.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

Hyperuricemia induces endothelial dysfunction and accelerates atherosclerosis by disturbing the asymmetric dimethylarginine/dimethylarginine dimethylaminotransferase 2 pathway.

Hyperuricemia is closely associated with the mobility and mortality of patients with cardiovascular diseases. However, how hyperuricemia accelerates atherosclerosis progression is not well understood. The balance between asymmetric dimethylarginine (ADMA) and dimethylarginine dimethylaminotransferases (DDAHs) is crucial to regulate vascular homeostasis. Therefore, we investigated the role of the ADMA/DDAH pathway in hyperuricemia-induced endothelial dysfunction and atherosclerosis and the underlying molecular mechanisms in endothelial cells (ECs) and apolipoprotein E-knockout (apoe-/-) mice. Our results demonstrated that uric acid at pathological concentrations increased the intracellular levels of ADMA and downregulated DDAH-2 expression without affecting DDAH-1 expression. Excess uric acid also reduced NO bioavailability and increased monocyte adhesion to ECs, which were abolished by using the antioxidant N-acetylcysteine, the nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin, or DDAH-2 overexpression. In apoe-/- mice, treatment with oxonic acid, a uricase inhibitor, increased the circulating level of uric acid, cholesterol, and lipid peroxidation; exacerbated systemic and aortic inflammation; and worsened atherosclerosis compared with vehicle-treated apoe-/- mice. Furthermore, oxonic acid-treated apoe-/- mice exhibited elevated ADMA plasma level and downregulated aortic expression of DDAH-2 protein. Notably, DDAH-2 overexpression in the ECs of apoe-/- mice prevented hyperuricemia-induced deleterious effects from influencing ADMA production, lipid peroxidation, inflammation, and atherosclerosis. Collectively, our findings suggest that hyperuricemia disturbs the balance of the ADMA/DDAH-2 axis, results in EC dysfunction, and, consequently, accelerates atherosclerosis.

Apigenin ameliorates hepatic lipid accumulation by activating the autophagy-mitochondria pathway.

Apigenin, a flavonoid isolated from plants, provides protection against non-alcoholic fatty liver disease. However, the mechanism by which apigenin decreases lipid accumulation in the liver is unclear. In this study, we investigated the molecular mechanism underlying the beneficial effect of apigenin on the hepatic deregulation of lipid metabolism. Oleic acid (OA)-induced lipid accumulation in human hepatoma cells (Huh7 cells) was used as an in vitro model. Western blot analysis was used for evaluating protein expression. Oil red O staining, Nile red staining, and conventional assay kits were used to assess the level of lipids. Immunocytochemistry was performed to observe mitochondrial morphology. Seahorse XF analyzer was used to measure mitochondrial bioenergetics. Treatment with OA induced lipid accumulation in Huh7 cells, which was attenuated by apigenin. Mechanistically, treatment with apigenin increased the expression of autophagy-related proteins including Beclin1, autophagy related gene 5 (ATG5), ATG7, and LC3II, and the formation of autophagolysosomes, leading to an increase in intracellular levels of fatty acids. Inhibition of autophagy by bafilomycin A1 or chloroquine abolished the protection of apigenin in OA-induced lipid accumulation. Apigenin up-regulated the protein expression related to the ß-oxidation pathway including acyl-CoA synthetase long chain family member 1, carnitine palmitoyltransferase 1α, acyl-CoA oxidase 1, peroxisome proliferator activated receptor (PPAR) α, and PPARγ coactivator 1-α. Moreover, apigenin increased the mitochondrial network structure and mitochondrial function by increasing the protein expression related to the process of mitochondria fusion and mitochondrial function. Collectively, our findings suggest that apigenin ameliorates hepatic lipid accumulation by activating the autophagy-mitochondrial pathway.

Di-(2-ethylhexyl) phthalate limits the pleiotropic effects of statins in chronic kidney disease patients undergoing dialysis and endothelial cells.

The level of di-(2-ethylhexyl) phthalate (DEHP) is elevated in chronic kidney disease patients undergoing dialysis. However, statins are unable to reduce the cardiovascular events in chronic dialysis patients. In this study, we investigated the effects of DEHP on statin-conferred pleiotropic effects and the underlying molecular mechanism in peritoneal dialysis (PD) patients and endothelial cells (ECs). In PD patients with serum DEHP level ≥0.0687 µg/mL, statin treatment was not associated with lower risk of cardiovascular disease. In ECs, exposure to DEHP abrogated the simvastatin-induced NO bioavailability and EC-related functions. Additionally, DEHP abolished the anti-inflammatory effect of simvastatin on the tumor necrosis factor α-induced upregulation of adhesion molecules and monocyte adhesion to ECs. Mechanistically, DEHP blunted the activation of transient receptor potential vanilloid type 1 (TRPV1), which is required for NO production by simvastatin in ECs. Notably, DEHP increased the activity and expression of protein phosphatase 2B (PP2B), a negative regulator of TRPV1 activity. The effect of DEHP on PP2B activation was mediated by the activation of the NADPH oxidase/reactive oxygen species (NOX-ROS) pathway. Inhibition of PP2B activity by pharmacological antagonists prevented the inhibitory effects of DEHP on simvastatin-induced Ca2+ influx, NO bioavailability, and EC migration, proliferation, tube formation, and anti-inflammatory action. Collectively, DEHP activates the NOX-ROS-PP2B pathway, which in turns inhibits TRPV1/Ca2+-dependent signaling and abrogates the statin-conferred pleiotropic protection in ECs.

Bromelain Confers Protection against the Non-Alcoholic Fatty Liver Disease in Male C57bl/6 Mice.

We aimed to investigate the effect of bromelain, the extract from stems of pineapples on the high-fat diet (HFD)-induced deregulation of hepatic lipid metabolism and non-alcoholic fatty liver disease (NAFLD), and its underlying mechanism in mice. Mice were daily administrated with HFD with or without bromelain (20 mg/kg) for 12 weeks, and we found that bromelain decreased the HFD-induced increase in body weight by ~30%, organ weight by ~20% in liver weight and ~40% in white adipose tissue weight. Additionally, bromelain attenuated HFD-induced hyperlipidemia by decreasing the serum level of total cholesterol by ~15% and triglycerides level by ~25% in mice. Moreover, hepatic lipid accumulation, particularly that of total cholesterol, free cholesterol, triglycerides, fatty acids, and glycerol, was decreased by 15-30% with bromelain treatment. Mechanistically, these beneficial effects of bromelain on HFD-induced hyperlipidemia and hepatic lipid accumulation may be attributed to the decreased fatty acid uptake and cholesteryl ester synthesis and the increased lipoprotein internalization, bile acid metabolism, cholesterol clearance, the assembly and secretion of very low-density lipoprotein, and the ß-oxidation of fatty acids by regulating the protein expression involved in the above mentioned hepatic metabolic pathways. Collectively, these findings suggest that bromelain has therapeutic value for treating NAFLD and metabolic diseases.

Endothelial Nitric Oxide Mediates the Anti-Atherosclerotic Action of Torenia concolor Lindley var. Formosama Yamazaki.

Torenia concolor Lindley var. formosama Yamazaki ethanolic extract (TCEE) is reported to have anti-inflammatory and anti-obesity properties. However, the effects of TCEE and its underlying mechanisms in the activation of endothelial nitric oxide synthase (eNOS) have not yet been investigated. Increasing the endothelium-derived nitric oxide (NO) production has been known to be beneficial against the development of cardiovascular diseases. In this study, we investigated the effect of TCEE on eNOS activation and NO-related endothelial function and inflammation by using an in vitro system. In endothelial cells (ECs), TCEE increased NO production in a concentration-dependent manner without affecting the expression of eNOS. In addition, TCEE increased the phosphorylation of eNOS at serine 635 residue (Ser635) and Ser1179, Akt at Ser473, calmodulin kinase II (CaMKII) at threonine residue 286 (Thr286), and AMP-activated protein kinase (AMPK) at Thr172. Moreover, TCEE-induced NO production, and EC proliferation, migration, and tube formation were diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Additionally, TCEE attenuated the tumor necrosis factor-α-induced inflammatory response as evidenced by the expression of adhesion molecules in ECs and monocyte adhesion onto ECs. These inflammatory effects of TCEE were abolished by L-NG-nitroarginine methyl ester (an NOS inhibitor). Moreover, chronic treatment with TCEE attenuated hyperlipidemia, systemic and aortic inflammatory response, and the atherosclerotic lesions in apolipoprotein E-deficient mice. Collectively, our findings suggest that TCEE may confer protection from atherosclerosis by preventing endothelial dysfunction.

Indoxyl sulfate impairs valsartan-induced neovascularization.

Studies revealed that the use of renin-angiotensin-aldosterone system antagonism is not associated with a statistically significant reduction in the risk of cardiovascular events in patients with chronic kidney disease (CKD) compared with that in the general population. We tested the hypothesis that indoxyl sulfate (IS) can interfere with the protective effect of valsartan-mediated on endothelial function in vitro and neovascularization in mice underwent subtotal nephrectomy. In human aortic endothelial cells, we first demonstrated that IS impaired the valsartan-mediated phosphorylation of eNOSThr495, nitric oxide production and tube formation via NADPH oxidase (NOX) and protein kinase C (PKC) phosphorylation, but this effect was suppressed by cotreatment with apocynin and calphostin C. In vivo, IS attenuated valsartan-induced angiogenesis in Matrigel plugs in mice. Moreover, in subtotal nephrectomy mice who underwent hindlimb ischemic surgery, valsartan significantly increased the mobilization of endothelial progenitor cells in circulation as well as the reperfusion of blood flow and density of CD31+ capillaries in ischemic limbs. However, IS attenuated the protective effect of valsartan-induced neovascularization and increased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Cotreatment of apocynin and calphostin C reversed the IS impaired-neovascularization and decreased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Our study suggests that the NOX/PKC/eNOS signaling pathway plays a pivotal role in the IS-mediated inhibition of valsartan-conferred beneficial effects on endothelial function in vitro and neovascularization in subtotal nephrectomy mice. We proposed a novel causative role for IS in cardiovascular complications in CKD patients.

The phosphatase activity of soluble epoxide hydrolase regulates ATP-binding cassette transporter-A1-dependent cholesterol efflux.

The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E-deficient mice, primarily in macrophage foam cells. Oxidized low-density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH-/- ) in macrophages markedly exacerbated oxLDL-induced lipid accumulation and decreased the expression of ATP-binding cassette transporters-A1 (ABCA1) and apolipoprotein AI-dependent cholesterol efflux following oxLDL treatment. The down-regulation of ABCA1 in sEH-/- macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH-ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL-induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL-induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.

Asymmetric Dimethylarginine Limits the Efficacy of Simvastatin Activating Endothelial Nitric Oxide Synthase.

BACKGROUND: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), is considered a risk factor for the pathogenesis of cardiovascular diseases. Simvastatin, a lipid-lowering drug with other pleiotropic effects, has been widely used for treatment of cardiovascular diseases. However, little is known about the effect and underlying molecular mechanisms of ADMA on the effectiveness of simvastatin in the vascular system. METHODS AND RESULTS: We conducted a prospective cohort study to enroll 648 consecutive patients with coronary artery disease for a follow-up period of 8 years. In patients with plasma ADMA level ≥0.49 µmol/L (a cut-off value from receiver operating characteristic curve), statin treatment had no significant effect on cardiovascular events. We also conducted randomized, controlled studies using in vitro and in vivo models. In endothelial cells, treatment with ADMA (≥0.5 µmol/L) impaired simvastatin-induced nitric oxide (NO) production, endothelial NO synthase (eNOS) phosphorylation, and angiogenesis. In parallel, ADMA markedly increased the activity of NADPH oxidase (NOX) and production of reactive oxygen species (ROS). The detrimental effects of ADMA on simvastatin-induced NO production and angiogenesis were abolished by the antioxidant, N-acetylcysteine, NOX inhibitor, or apocynin or overexpression of dimethylarginine dimethylaminohydrolase 2 (DDAH-2). Moreover, in vivo, ADMA administration reduced Matrigel plug angiogenesis in wild-type mice and decreased simvastatin-induced eNOS phosphorylation in aortas of apolipoprotein E-deficient mice, but not endothelial DDAH-2-overexpressed aortas. CONCLUSIONS: We conclude that ADMA may trigger NOX-ROS signaling, which leads to restricting the simvastatin-conferred protection of eNOS activation, NO production, and angiogenesis as well as the clinical outcome of cardiovascular events.

Transient receptor potential vanilloid type 1 is vital for (-)-epigallocatechin-3-gallate mediated activation of endothelial nitric oxide synthase.

SCOPE: Epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, has beneficial effects on physiological functions of endothelial cells (ECs), yet the detailed mechanisms are not fully understood. In this study, we investigated the role of transient receptor potential vanilloid type 1 (TRPV1), a ligand-gated nonselective calcium channel, in EGCG-mediated endothelial nitric oxide (NO) synthase (eNOS) activation and angiogenesis. METHODS AND RESULTS: In ECs, treatment with EGCG time-dependently increased the intracellular level of Ca(2+) . Removal of extracellular calcium (Ca(2+) ) by EGTA or EDTA or inhibition of TRPV1 by capsazepine or SB366791 abrogated EGCG-increased intracellular Ca(2+) level in ECs or TRPV1-transfected HEK293 cells. Additionally, EGCG increased the phsophorylation of eNOS at Ser635 and Ser1179, Akt at Ser473, calmodulin-dependent protein kinase II (CaMKII) at Thr286 and AMP-activated protein kinase (AMPK) at Thr172, all abolished by the TRPV1 antagonist capsazepine. EGCG-induced NO production was diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Moreover, blocking TRPV1 activation prevented EGCG-induced EC proliferation, migration, and tube formation, as well as angiogenesis in Matrigel plugs in mice. CONCLUSION: EGCG may trigger activation of TRPV1-Ca(2+) signaling, which leads to phosphorylation of Akt, AMPK, and CaMKII; eNOS activation; NO production; and, ultimately, angiogenesis in ECs.