Interfacial Bond-Slip Model for CFRP Plate Externally Bonded to Corroded Steel Plate.

The purpose of this study is to establish the interfacial bond-slip model for CFRP plate externally bonded to corroded steel plate. The present bond-slip models for CFRP materials bonded to uncorroded steel plate were first reviewed. Thirty-four double-lap joints were tested to investigate the effect of corrosion duration and adhesive thickness on the bond behavior between CFRP plates and corroded steel plates, and the bond-slip curves for the bonding interface with different adhesive thickness and corrosion duration were obtained combined with the CFRP plate strain distribution data. A new bond-slip model for CFRP plate externally bonded to corroded steel plate was proposed, and the expression of the characteristic parameters, which included the maximum bond resistance τf, the relative slip at the peak bond stress s1, the fitting parameter α, and the interfacial fracture energy Gf, were also developed based on the careful regression analysis of the present data. The influence of the corrosion duration and construction adhesive thickness on the bond-slip relationship were accounted together and expressed as a new parameter; that is, the effective adhesive thickness teff. The comparison between the predicted values and experimental results indicated that the proposed bond-slip model can be applied to reproduce the structural response of the CFRP plate-corroded steel plate double-lap joint with reasonable accuracy. The outcome of this study can provide meaningful references and essential data for the reliable application of CFRP strengthening systems in the performance improvement of corroded steel structures.

Deterioration of Mechanical Properties and the Damage Constitutive Model of Corroded Steel in an Industrial Environment.

To investigate the degradation law of the mechanical properties of corroded steel, the standard specimens from machining steel members in service for 9 years in an industrial environment were subjected to tensile tests. The influences of different degrees and types of corrosion on the fracture path, stress-strain curve, and mechanical properties of specimens were discussed. Finally, the damage constitutive model of corroded steel was established based on the damage mechanics theory. The results showed that the failure modes of corroded specimens were related to the degrees and types of corrosion. The fracture morphology of specimens with general corrosion were step-like and the fractures of steel were uneven. However, those with local corrosion were mainly flat-like, and the fracture path was along the cross section where the larger corrosion pits were located. The fracture path of the specimen was related to the interaction of the corroded surface and internal material defects (holes). Meanwhile, with the increase of corrosion degree, the yield platform of stress-strain curve gradually became shorter, or even disappeared, and the ultimate strain and elongation at break decreased, implying that the ductility of steel became worse. Ultimately, the good agreement between the curves of the model and test indicated that the damage model could reflect the damage development process of corroded steel in the tensile process better. Corrosion damage resulted in the decrease in the damage threshold, and the damage variable D decreased by the time fracture occurred and the maximum reduction rate was up to 62.5%.

Fatigue Crack Propagation Prediction of Corroded Steel Plate Strengthened with Carbon Fiber Reinforced Polymer (CFRP) Plates.

The purpose of this study is to investigate the mechanism of improving fatigue performance and the estimation model of fatigue life for corroded steel plate strengthened with CFRP plates. A new two-stage fatigue crack propagation prediction model for the corroded steel plate strengthened with CFRP plates was proposed; moreover, the identification of critical rust pits and the equivalent method of initial cracks, and the calculation method of stress intensity factor (SIF) values at the crack tip were established. The accuracy of the proposed model was verified by comparing the predicted and tested fatigue life of the corroded steel plate strengthened with CFRP plates. Finally, the proposed two-stage crack propagation model was applied to carry out a parameter analysis to investigate the effect of weight loss rate, equivalent initial crack size, adhesive thickness, CFRP stiffness and CFRP prestress level on the fatigue crack propagation of the corroded steel plate strengthened with CFRP plates. Results showed that the maximum depth and the average width of the rust pits were suggested to be taken as the equivalent dimensions of the initial semi-elliptical surface crack for the fatigue crack propagation prediction of corroded steel plate strengthened with CFRP plates. Increasing the weight loss rate of the corroded steel plate, the initial crack size or the adhesive thickness would accelerate the crack growth and reduce the fatigue life, whereas increasing the stiffness or prestress level of the CFRP plate would significantly reduce the crack growth rate and increase the fatigue life. The smaller the initial crack size, the more sensitive the crack propagation life was to the variation of equivalent initial crack size. The influence of adhesive thickness on the fatigue life was limited and convergent, and the application of prestressing could significantly improve the utilization rate of CFRP materials and the fatigue strengthening effect of the corroded steel plate.

Stress Concentration Analysis of the Corroded Steel Plate Strengthened with Carbon Fiber Reinforced Polymer (CFRP) Plates.

The purpose of this study is to investigate the stress concentration of a corroded steel plate strengthened with carbon fiber reinforced polymer (CFRP) plates. An accelerated corrosion experiment was first executed to acquire corroded steel plates, and then surface profile measurements were conducted to obtain 3D coordinate data of the corroded steel surface. Finite element models considering the surface morphology of the corroded steel plate and the interfacial bonding properties between the CFRP plate and the corroded steel plate were established to investigate the stress concentration of the corroded steel plate strengthened with and without CFRP plates. The reliability of the numerical modeling method was verified based on a mesh convergence analysis and a comparison of the fatigue test, 3D morphology scanning, and numerical analysis results. Specimens with five levels of corrosion damage, six kinds of CFRP-strengthening stiffness, five kinds of adhesive thickness, and five levels of CFRP prestress were numerically modeled. The primary indications consist of features of stress distribution, and the stress concentration factors Kt and Ktg were analyzed. Results showed that the features of stress distribution and the stress concentration factor Kt of the corroded steel plate strengthened with and without CFRP plates are only related to the shape, size, and position of rust pits, but not to the degree of uniform corrosion or the reinforcement parameters. The value of Kt for the corroded steel plate with a corrosion duration of 6~18 months and a weight loss rate of 9.16~21.78% was approximately 1.199~1.345. The converted stress concentration factor Ktg has more practical significance than the stress concentration factor Kt in describing the influence of corrosion and CFRP reinforcement on the peak tensile stress of the corroded steel plate. The value of Ktg increased linearly with the increase of the weight loss rate of the corroded steel plate and decreased appreciably with the increase of the strengthening stiffness and prestress level of the CFRP plates, and it presented a very small increasing trend with the increase of the adhesive thickness.

Estimation of Bond Strength and Effective Bond Length for the Double Strap Joint between Carbon Fiber Reinforced Polymer (CFRP) Plate and Corroded Steel Plate.

In this paper, we examine the development of the estimation models of bond strength and effective bond length for a double strap joint between carbon fiber reinforced polymer (CFRP) plate and corroded steel plate. The experimental study on the bond behavior between CFRP plate and corroded steel plate is summarized first and the analytical interfacial bond-slip model for CFRP plate externally bonded to corroded steel plate is proposed. Based on the theoretical stress analysis for the CFRP plate-corroded steel plate double-lap joint, the piecewise expressions of the interfacial shear stress and the normal peel stress of the interface between CFRP plate and corroded steel plate were established. The estimation models of the bond strength and the effective bond length for the double strap joint between the CFRP plate and the corroded steel plate were consequently developed on the basis of interfacial stress distribution equations and the stress boundary conditions. The comparison between the predicted and experimental results indicated that the proposed models could be adopted to predict the bond strength and effective bond length for the CFRP plates externally bonded to corroded steel substrates with reasonable accuracy. The proposed estimation models are expected to provide meaningful references and essential data for the reliable application of CFRP strengthening system to the performance improvement of corroded steel structures.

Oxidative stress and calcium dysregulation by palmitate in type 2 diabetes.

Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca2+ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca2+ release and thereby deplete ER Ca2+ stores. The resulting ER Ca2+ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca2+ release, cytosolic and mitochondrial matrix Ca2+ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca2+ depletion mediate persistent Ca2+ influx, further impairing cytosolic and mitochondrial Ca2+ homeostasis. Mitochondrial Ca2+ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in ß-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.

Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high-phosphate-induced mitochondrial oxidative stress and defective insulin secretion.

Elevated plasma levels of inorganic phosphate (Pi) are harmful, causing, among other complications, vascular calcification and defective insulin secretion. The underlying molecular mechanisms of these complications remain poorly understood. We demonstrated the role of Pi transport across the plasmalemma on Pi toxicity in INS-1E rat clonal ß cells and rat pancreatic islet cells. Type III sodium-phosphate cotransporters (NaPis) are the predominant Pi transporters expressed in insulin-secreting cells. Transcript and protein levels of sodium-dependent phosphate transporter 1 and 2 (PiT-1 and -2), isotypes of type III NaPi, were up-regulated by high-Pi incubation. In patch-clamp experiments, extracellular Pi elicited a Na+-dependent, inwardly rectifying current, which was markedly reduced under acidic extracellular conditions. Cellular uptake of Pi elicited cytosolic alkalinization; intriguingly, this pH change facilitated Pi transport into the mitochondrial matrix. Increased mitochondrial Pi uptake accelerated superoxide generation, mitochondrial permeability transition (mPT), and endoplasmic reticulum stress-mediated translational attenuation, leading to reduced insulin content and impaired glucose-stimulated insulin secretion. Silencing of PiT-1/2 prevented Pi-induced superoxide generation and mPT, and restored insulin secretion. We propose that Pi transport across the plasma membrane and consequent cytosolic alkalinization could be a therapeutic target for protection from Pi toxicity in insulin-secreting cells, as well as in other cell types.-Nguyen, T. T., Quan, X., Xu, S., Das, R., Cha, S.-K., Kong, I. D., Shong, M., Wollheim, C. B., Park, K.-S. Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high-phosphate-induced mitochondrial oxidative stress and defective insulin secretion.

Autocrine insulin increases plasma membrane K(ATP) channel via PI3K-VAMP2 pathway in MIN6 cells.

Regulation of ATP-sensitive inwardly rectifying potassium (KATP) channel plays a critical role in metabolism-secretion coupling of pancreatic ß-cells. Released insulin from ß-cells inhibits insulin and glucagon secretion with autocrine and paracrine modes. However, molecular mechanism by which insulin inhibits hormone secretion remains elusive. Here, we investigated the effect of autocrine insulin on surface abundance of KATP channel in mouse clonal ß-cell line, MIN6. High glucose increased plasmalemmal sulfonylurea receptor 1 (SUR1), a component of KATP channel as well as exogenous insulin treatment. SUR1 trafficking by high glucose or insulin was blocked by inhibition of phosphoinositide 3-kinase (PI3K) with wortmannin. Pretreatment with brefeldin A or silencing of vesicle-associated membrane protein 2 (VAMP2) abolished insulin-mediated upregulation of surface SUR1. Functionally, glucose-stimulated cytosolic Ca(2+) ([Ca(2+)]i) increase was blunted by insulin or diazoxide, a KATP channel opener. Insulin-induced suppression of [Ca(2+)]i oscillation was prevented by an insulin receptor blocker. These results provide a novel molecular mechanism for autocrine negative feedback regulation of insulin secretion.

Transforming Growth Factor ß1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis.

TGF-ß is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-ß1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-ß1-induced apoptosis in immortalized podocytes. TGF-ß1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-ß receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTORΔ augmented TGF-ß1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-ß1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-ß1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-ß1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-ß1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-ß1-induced ERK1/2-mTORactivation. Our data suggest that TGF-ß1 increases translation of Nox4 through the Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic kidney diseases.

Mitochondrial oxidative stress mediates high-phosphate-induced secretory defects and apoptosis in insulin-secreting cells.

Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal ß-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation, and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ΔΨm hyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.

Gallic acid regulates body weight and glucose homeostasis through AMPK activation.

Gallic acid [3,4,5-trihydroxybenzoic acid (GA)], a natural phytochemical, is known to have a variety of cellular functions including beneficial effects on metabolic syndromes. However, the molecular mechanism by which GA exerts its beneficial effects is not known. Here we report that GA plays its role through the activation of AMP-activated protein kinase (AMPK) and by regulating mitochondrial function via the activation of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Sirtuin 1 (Sirt1) knockdown significantly blunted GA's effect on PGC1α activation and downstream genes, suggesting a critical role of the AMPK/Sirt1/PGC1α pathway in GA's action. Moreover, diet-induced obese mice treated with GA showed significantly improved glucose and insulin homeostasis. In addition, the administration of GA protected diet-induced body weight gain without a change in food intake. Biochemical analyses revealed a marked activation of AMPK in the liver, muscle, and interscapular brown adipose tissue of the GA-treated mice. Moreover, uncoupling protein 1 together with other genes related to energy expenditure was significantly elevated in the interscapular brown adipose tissue. Taken together, these results indicate that GA plays its beneficial metabolic roles by activating the AMPK/Sirt1/PGC1α pathway and by changing the interscapular brown adipose tissue genes related to thermogenesis. Our study points out that targeting the activation of the AMPK/Sirt1/PGC1α pathway by GA or its derivatives might be a potential therapeutic intervention for insulin resistance in metabolic diseases.

Essential role of mitochondrial Ca2+ uniporter in the generation of mitochondrial pH gradient and metabolism-secretion coupling in insulin-releasing cells.

In pancreatic ß-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca(2+) influx, which triggers insulin exocytosis. The mitochondrial Ca(2+) uniporter (MCU) mediates Ca(2+) uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal ß-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca(2+) rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca(2+). Suppression of the putative Ca(2+)/H(+) antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca(2+)-induced matrix acidification. These results demonstrate that MCU-mediated Ca(2+) uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.

Different spectroscopic and molecular modeling studies on the interaction between cyanidin-3-O-glucoside and bovine serum albumin.

Anthocyanin is one of the flavonoid phytopigments that shows strong antioxidant activity. The cyanidin-3-O-glucoside (C3G) is one of the principal types of anthocyanins. To understand the interaction between C3G and bovine serum albumin (BSA), fluorescence spectroscopy, ultraviolet-visible absorption, Fourier transform infrared spectroscopy, circular dichroism and molecular modeling techniques were used. Binding constant (K(a)) and the number of binding sites (n) were calculated. The quenching mechanism of fluorescence of BSA by C3G was discussed. The results studied by Fourier transform infrared spectroscopy and circular dichroism experiments indicate that the secondary structures of the protein have been changed by the interaction of C3G with BSA. The result of molecular modeling confirmed that the C3G bound to the site I (sub-domain IIA) of BSA, and that the hydroxyl groups in the B ring of C3G took part in the binding with BSA.

Upregulation of mitochondrial Nox4 mediates TGF-ß-induced apoptosis in cultured mouse podocytes.

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-ß in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-ß in immortalized mouse podocytes. Exogenous TGF-ß1-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-ß1 markedly depolarized mitochondrial membrane potential. TGF-ß1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-ß receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-ß1. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-ß1. These results suggest that TGF-ß1-induced mitochondrial Nox4 upregulation via the TGF-ß receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.

Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells.

Here, we have investigated the role of inorganic phosphate (Pi) transport in mitochondria of rat clonal ß-cells. In α-toxin-permeabilized INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and Pi. In the presence of substrates, addition of Pi caused mitochondrial matrix acidification and hyperpolarisation which promoted ATP export. Dissipation of the mitochondrial pH gradient or pharmacological inhibition of Pi transport blocked the effects of Pi on electrochemical gradient and ATP export. Knock-down of the phosphate transporter PiC, however, neither prevented Pi-induced mitochondrial activation nor glucose-induced insulin secretion. Using (31)P NMR we observed reduction of Pi pools during nutrient stimulation of INS-1E cells. Interestingly, Pi loss was less pronounced in mitochondria than in the cytosol. We conclude that matrix alkalinisation is necessary to maintain a mitochondrial Pi pool, at levels sufficient to stimulate energy metabolism in insulin-secreting cells beyond its role as a substrate for ATP synthesis.

High-dose clevudine impairs mitochondrial function and glucose-stimulated insulin secretion in INS-1E cells.

BACKGROUND: Clevudine is a nucleoside analog reverse transcriptase inhibitor that exhibits potent antiviral activity against hepatitis B virus (HBV) without serious side effects. However, mitochondrial myopathy has been observed in patients with chronic HBV infection taking clevudine. Moreover, the development of diabetes was recently reported in patients receiving long-term treatment with clevudine. In this study, we investigated the effects of clevudine on mitochondrial function and insulin release in a rat clonal ß-cell line, INS-1E. METHODS: The mitochondrial DNA (mtDNA) copy number and the mRNA levels were measured by using quantitative PCR. MTT analysis, ATP/lactate measurements, and insulin assay were performed. RESULTS: Both INS-1E cells and HepG2 cells, which originated from human hepatoma, showed dose-dependent decreases in mtDNA copy number and cytochrome c oxidase-1 (Cox-1) mRNA level following culture with clevudine (10 µM-1 mM) for 4 weeks. INS-1E cells treated with clevudine had reduced total mitochondrial activities, lower cytosolic ATP contents, enhanced lactate production, and more lipid accumulation. Insulin release in response to glucose application was markedly decreased in clevudine-treated INS-1E cells, which might be a consequence of mitochondrial dysfunction. CONCLUSIONS: Our data suggest that high-dose treatment with clevudine induces mitochondrial defects associated with mtDNA depletion and impairs glucose-stimulated insulin secretion in insulin-releasing cells. These findings partly explain the development of diabetes in patients receiving clevudine who might have a high susceptibility to mitochondrial toxicity.