The neuroprotective role of rosiglitazone in advanced glycation end product treated human neural stem cells is PPARgamma-dependent

Hyperglycemia is accompanied by an accelerated formation rate of advanced glycation end products (AGEs), which is associated with the pathogenesis of diabetic neuronal deficits. Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors and its ligands are known to control many physiological, pathological and inflammatory pathways. Weinvestigated the hypothesis that the PPARγ agonist (rosiglitazone) would abrogate AGEs-mediated neurotoxic effects on human neural stem cells (hNSCs), by whichAGEs may play a role in diabetic-related neuronal impairment. Here, we show that rosiglitazone treatment increases cell viability of hNSCs via downregulation of caspase 3 activity. These rescue effects were extended in our studies showingrosiglitazone-mediated activation of PPARγ reversed the expression levels of two neuroprotective factors (Bcl-2 and PGC1α) that were downregulated in hNSCs exposed to AGEs alone. The stimulation of mitochondrial function and anti-oxidative stress by rosiglitazone was associated with activation of the PGC1α pathway by up-regulation of mitochondrial (NRF-1 and Tfam) and oxidative defense (SOD1, SOD2 and Gpx1) genes. Moreover, rosiglitazone significantly normalized the inflammatory responses (TNF-α and IL-1β), NF-κB (p65), and inflammatory genes (iNOS and COX-2) in the hNSCs treated with AGEs. This neuroprotective effect of rosiglitazone was effectively blocked by PPARγ-specific antagonist (GW9662), demonstrating that the action of rosiglitazone was mediated by at PPARγ-dependent pathway. Collectively, these novel findings show AGEs induce neurotoxic effects in hNSCs, and provide important mechanistic insights that may explain the increased risk of neuronal impairment deficits in diabetic patients. More importantly, these data show rosiglitazone-mediated activation of PPARγ-dependent signaling is neuroprotective in AGE-treated hNSCs, and suggests PPARγ ligands may be useful in the therapeutic management of patients with neurodegenerative diseases     

Inducible nitric oxide synthase and cyclooxgenase-2 mediate protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

The induction of inducible nitric oxide synthase (iNOS) in response to different stress is associated with simultaneous induction of cyclooxygenase-2 (COX-2) in various cell types. Both iNOS and COX-2 have been reported to mediate the late phase of cardioprotection induced by different preconditioning. However, whether both iNOS and COX-2 are mediators in the neuroprotection induced by preconditioning with hydrogen peroxide (H(2)O(2)) at low concentration is unknown. In this study, using the neurosecretory cell line-PC12 cells to set up the model of neuroprotection of preconditioning with H(2)O(2) against apoptosis, we first investigate what changes in expression of iNOS and COX-2 appear during H(2)O(2) preconditioning, then determine if both iNOS inhibitor and COX-2 inhibitor interfere with the neuroprotection elicited by preconditioning with H(2)O(2). We found that preconditioning with H(2)O(2) at 10 microM significantly protected PC12 cells against apoptosis induced by lethal H(2)O(2) (50 microM) and increased the expression of iNOS and COX-2 and that selective iNOS inhibitor, aminoguanidine (AG) and COX-2 inhibitor, NS-398 obviously blocked the protective effects induced by preconditioning with 10 microM H(2)O(2). The results of this study suggest that both iNOS and COX-2 are mediators of the neuroprotection induced by preconditioning with oxidative stress (H(2)O(2) at low concentration) in PC12 cells.     

Advanced Glycation End Products Induce Human Corneal Epithelial Cells Apoptosis through Generation of Reactive Oxygen Species and Activation of JNK and p38 MAPK Pathways

Advanced Glycation End Products (AGEs) has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS) and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA) induced Human telomerase-immortalized corneal epithelial cells (HUCLs) apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE). AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC) or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.     

Rosiglitazone Suppresses Glioma Cell Growth and Cell Cycle by Blocking the Transforming Growth Factor-Beta Mediated Pathway

Glioma is one of the most malignant tumors in the central nervous system. As a peroxisome proliferator-activated receptor γ (PPAR-γ) activator, the thiazolidinediones (TZDs) induce growth arrest and cell death in a broad spectrum of tumor cells. In this study, we investigated the role of rosiglitazone in glioma cells. We found that rosiglitazone, a member of TZDs, suppresses growth of human glioma cell lines U87 and U251. Rosiglitazone also induces cell cycle arrest and apoptosis, which may be the mechanism of its anti-proliferation effect. Next, we found that rosiglitazone suppresses the expression of TGF-beta and its receptor TGF-betaR2, and suppresses phosphorylation of Smad3. Rosiglitazone also inhibits formation of the Smad3/Smad4 complex. Furthermore, Rosiglitazone affects the expression of Smad3/Smad4 associated regulators of gene expression, including p21 and c-Myc. These results suggest that rosiglitazone suppresses growth and cell cycle of human glioma cells by blocking the TGF-beta mediated pathway.     

Thiazolidinediones inhibit the growth of PC12 cells both in vitro and in vivo

Thiazolidinediones (TZDs) have recently been proposed as a therapy for PPARγ-expressing tumors. Pheochromocytoma (PHEO) is associated with high morbidity and mortality due to excess catecholamine production, and few effective drug therapies currently exist. We investigated the effects of TZDs on PHEO both in vitro and in vivo. PPARγ protein was expressed in human adrenal PHEO tissues as well as in rat PHEO cells, PC12. TZDs, including rosiglitazone (RGZ) and pioglitazone (PGZ), inhibited proliferation of PC12 cells in a dose-dependent manner and increased casapse-3 expression of PC12 cells. TZDs also reduced expression of cyclin E and cyclin-dependent kinase2. RGZ inhibited nerve growth factor-induced neurite outgrowth and reduced expression of catecholamine-synthesizing enzymes. Finally, rat PHEO growth generated by subcutaneous injection of PC12 cells was slowed in an RGZ-treated mouse. These data suggest that TZDs may be a promising therapeutic approach for medical treatment for PHEO.     

Brain PPAR-γ promotes obesity and is required for the insulin-sensitizing effect of thiazolidinediones

In adipose tissue, muscle, liver and macrophages, signaling by the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ) is a determinant of insulin sensitivity and this receptor mediates the insulin-sensitizing effects of thiazolidinediones (TZDs). As PPAR-γ is also expressed in neurons, we generated mice with neuron-specific Pparg knockout (Pparg brain knockout (BKO)) to determine whether neuronal PPAR-γ signaling contributes to either weight gain or insulin sensitivity. During high-fat diet (HFD) feeding, food intake was reduced and energy expenditure increased in Pparg-BKO mice compared to Pparg(f/f) mice, resulting in reduced weight gain. Pparg-BKO mice also responded better to leptin administration than Pparg(f/f) mice. When treated with the TZD rosiglitazone, Pparg-BKO mice were resistant to rosiglitazone-induced hyperphagia and weight gain and, relative to rosiglitazone-treated Pparg(f/f) mice, experienced only a marginal improvement in glucose metabolism. Hyperinsulinemic euglycemic clamp studies showed that the increase in hepatic insulin sensitivity induced by rosiglitazone treatment during HFD feeding was completely abolished in Pparg-BKO mice, an effect associated with the failure of rosiglitazone to improve liver insulin receptor signal transduction. We conclude that excess weight gain induced by HFD feeding depends in part on the effect of neuronal PPAR-γ signaling to limit thermogenesis and increase food intake. Neuronal PPAR-γ signaling is also required for the hepatic insulin sensitizing effects of TZDs.     

Advanced glycosylation end products induce nitric oxide synthase expression in C6 glioma cells: involvement of a p38 MAP kinase-dependent mechanism.

The mitogen-activated protein kinase (MAPK) pathway is believed to function as an important mediator of inducible nitric oxide synthase (iNOS) expression. In the present study, we investigated the role of the p38 MAPK signaling pathway in advanced glycosylation end products (AGEs)-induced iNOS expression in C6 glioma cells. AGEs caused a dose-dependent increase of nitrite accumulation in C6 glioma cells. The AGEs-stimulated nitrite production from C6 glioma cells was inhibited by actinomycin D, cyclohexamide, and the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), suggesting that the increase of AGEs-induced nitrite release is due to iNOS up-regulation. Consistently, treatment of C6 glioma cells with AGEs induced iNOS protein expression. AGEs-stimulated nitrite production was inhibited by pretreatment of C6 glioma cells with anti-AGEs antibodies (1:100 or 1:50). The tyrosine kinase inhibitor (genistein and tyrphostin), the Ras-farnesyl transferase inhibitor (FPT inhibitor-II), or the p38 MAPK inhibitor (SB203580) suppressed AGEs-induced iNOS expression and nitrite release from C6 glioma cells. AGEs activated p38 MAPK in C6 glioma cells, and this effect was blocked by genistein (20 microM), tyrphostin (30 microM), FPT inhibitor-II (20 microM), and SB203580 (10 microM). Taken together, our data suggest that AGEs may activate the pathways of tyrosine kinase and Ras to induce p38 MAPK activation, which in turn induces iNOS expression and NO production in C6 glioma cells.     

Advanced glycation endproducts influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts

Glycation reactions resulting in the generation and accumulation of advanced glycation endproducts (AGEs) are potential mechanisms by which bone protein may be altered in vivo. AGEs accumulate in the bone increasingly with age come into close contact with osteoblasts or osteoclasts. The direct effect of AGEs on bone cells has not been thoroughly investigated. This study aimed to examine whether glycated bovine serum albumin (AGE - BSA) as an AGE modulate the mRNA expression of various genes in primary human osteoblast cultures. The following parameters were included: RAGE (receptor for AGEs), alkaline phosphatase, osteocalcin, osterix and RANKL (receptor activator of nuclear factor-kappaB ligand). Primary human osteoblast cultures were obtained from bone specimens of six patients with osteoarthrosis. Human osteoblasts were treated in AGE - BSA or control-BSA (non-glycated BSA) containing medium (5 mg/ml each) over a time course of seven days. After RT-PCR the mRNA expression was measured by real-time PCR. Related to control - BSA exposure, the mRNA expression of RAGE, RANKL and osterix increased during AGE - BSA treament. For alkaline phosphatase and osteocalcin a tendency of down-regulation was found. In summary, the study presents evidence that advanced glycation end products accumulated in bone alter osteoblasts by activation the AGE - RAGE pathway (RAGE mRNA up-regulation), inducing enhanced osteoclastogenesis (RANKL mRNA up-regulation) and impaired matrix mineralization (down-regulation of alkaline phosphatase and osteocalcin mRNA). Thus, AGEs may play a functional role in the development of bone diseases (e.g. osteoporosis).     

Down-regulation of lipoprotein lipase increases glucose uptake in L6 muscle cells

Thiazolidinediones (TZDs) are synthetic hypoglycemic agents used to treat type 2 diabetes. TZDs target the peroxisome proliferator activated receptor-gamma (PPAR-γ) and improve systemic insulin sensitivity. The contributions of specific tissues to TZD action, or the downstream effects of PPAR-γ activation, are not very clear. We have used a rat skeletal muscle cell line (L6 cells) to demonstrate that TZDs directly target PPAR-γ in muscle cells. TZD treatment resulted in a significant repression of lipoprotein lipase (LPL) expression in L6 cells. This repression correlated with an increase in glucose uptake. Down-regulation of LPL message and protein levels using siRNA resulted in a similar increase in insulin-dependent glucose uptake. Thus, LPL down-regulation improved insulin sensitivity independent of TZDs. This finding provides a novel method for the management of insulin resistance.     

Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

Thiazolidinediones, specific peroxisome proliferator-activated receptor-?γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.     

Microglial cell death induced by glycated bovine serum albumin: nitric oxide involvement

Nonenzymatic glycation results in the formation of advanced glycation end products (AGEs) through a nonenzymatic multistep reaction of reducing sugars with proteins. AGEs have been suspected to be involved in the pathogenesis of several chronic clinical neurodegenerative complications including Alzheimer's disease, which is characterized with the activation of microglial cells in neuritic plaques. To find out the consequence of this activation on microglial cells, we treated the cultured microglial cells with different glycation levels of Bovine Serum Albumin (BSA) which were prepared in vitro. Extent of glycation of protein has been characterized during 16 weeks of incubation with glucose. Treatment of microglial cells with various levels of glycated albumin induced nitric oxide (NO) production and consequently cell death. We also tried to find out the mode of death in AGE-activated microglial cells. Altogether, our results suggest that AGE treatment causes microglia to undergo NO-mediated apoptotic and necrotic cell death in short term and long term, respectively. NO production is a consequence of iNOS expression in a JNK dependent RAGE signalling after activation of RAGE by AGE-BSA.     

Inhibition of ROS-Activated p38MAPK Pathway is Involved in the Protective Effect of H2S Against Chemical Hypoxia-Induced Inflammation in PC12 Cells

We have demonstrated the neuroprotection of hydrogen sulfide (H₂S) against chemical hypoxia-induced injury by inhibiting p38MAPK pathway. The present study attempts to evaluate the effect of H₂S on chemical hypoxia-induced inflammation responses and its mechanisms in PC12 cells. We found that treatment of PC12 cells with cobalt chloride (CoCl₂, a hypoxia mimetic agent) enhanced IL-6 secretion, nitric oxide (NO) generation and expression levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). L-canavanine, a selective iNOS inhibitor, partly blocked CoCl₂-induced cytotoxicity, apoptosis and mitochondrial insult. In addition, 7-Nitroindazole (7-NI), an inhibitor of nNOS, also partly attenuated the CoCl₂-induced cytotoxicity. The inhibition of p38MAPK by SB203580 (a selective p38MAPK inhibitor) or genetic silencing of p38MAPK by RNAi (Si-p38) depressed not only CoCl₂-induced iNOS expression, NO production, but also IL-6 secretion. In addition, N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, conferred a similar protective effect of SB203580 or Si-p38 against CoCl₂-induced inflammatory responses. Importantly, pretreatment of PC12 cells with exogenous application of sodium hydrosulfide (a H₂S donor, 400 μmol/l) for 30 min before exposure to CoCl₂ markedly attenuated chemical hypoxia-stimulated iNOS and nNOS expression, NO generation and IL-6 secretion as well as p38MAPK phosphorylation in PC12 cells. Taken together, we demonstrated that p38MAPK-iNOS pathway contributes to chemical hypoxia-induced inflammation and that H₂S produces an anti-inflammatory effect in chemical hypoxia-stimulated PC12 cells, which may be partly due to inhibition of ROS-activated p38MAPK-iNOS pathway.     

Glycation-induced modification of tissue-specific ECM proteins: A pathophysiological mechanism in degenerative diseases

BackgroundGlycation driven generation of advanced glycation end products (AGEs) and their patho-physiological role in human degenerative diseases has remained one of the thrust areas in the mainstream of disease biology. Glycation of extracellular matrix (ECM) proteins have deleterious effect on the mechanical and functional properties of tissues. Owing to the adverse pathophysiological concerns of glycation, there is a need to decipher the underlying mechanisms.Scope of reviewAGE-modified ECM proteins affect the cell in the vicinity by altering protein structure-function, matrix-matrix or matrix-cell interaction and by activating signalling pathway through receptor for AGE. This review is intended for addressing the AGE-induced modification of tissue-specific ECM proteins and its implication in the pathogenesis of various organ-specific human ailments.Major conclusionsThe glycation affects the canonical cell behaviour due to alteration in the interaction of glycated ECM with receptors like integrins and discodin domain, and the signalling cues generated subsequently affect the downstream signalling pathways. Consequently, the variation of structural and functional properties of tissues due to matrix glycation helps in the initiation or progression of the disease condition.General significanceThis review offers comprehensive knowledge about the remodelling of glycation induced ECM and tissue-specific pathological concerns. As glycation of ECM affects the normal tissues and cell behaviour, the scientific discourse may also provide cues for developing candidate drugs that may help in attenuating the adverse effects of AGEs and perhaps open a research window of tailoring novel strategies for the management of glycation induced human degenerative diseases.     

Delayed adenosine A1 receptor preconditioning in rat myocardium is MAPK dependent but iNOS independent

Adenosine A1 receptor delayed preconditioning (PC) against myocardial infarction has been well described; however, there have been limited investigations of the signaling mechanisms that mediate this phenomenon. In addition, there are multiple conflicting reports on the role of inducible nitric oxide synthase (iNOS) in mediating A1 late-phase PC. The purpose of this study was to determine the roles of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) in in vivo delayed A1 receptor PC and whether this protection at the myocyte level is due to upregulation of iNOS. Myocardial infarct size was measured in open-chest anesthetized rats 24 h after treatment with vehicle or the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 100 microg/kg ip). Additional rats receiving CCPA were pretreated with the p38 inhibitor SB-203580 (1 mg/kg ip) or the MAPK/ERK kinase (MEK) inhibitor PD-098059 (0.5 mg/kg ip). At 24 h after CCPA administration, a group of animals was given the iNOS inhibitor 1400 W 10 min before ischemia. Treatment with CCPA reduced infarct size from 48 +/- 2 to 28 +/- 2% of the area at risk, an effect that was blocked by both SB-203580 and PD-098059 but not 1400 W. Ventricular myocytes isolated 24 h after CCPA injection exhibited significantly reduced oxidative stress during H2O2 exposure compared with myocytes from vehicle-injected animals, and this effect was not blocked by the iNOS inhibitor 1400 W. Western blot analysis of whole heart and cardiac myocyte protein samples revealed no expression of iNOS 6 or 24 h after CCPA treatment. These results indicate that adenosine A1 receptor delayed PC in rats is mediated by MAPK-dependent mechanisms, but this phenomenon is not associated with the early or late expression of iNOS.     

Comparison of bovine serum albumin glycation by ribose and fructose in vitro and in vivo

Advanced glycation end products (AGEs) play a critical pathogenic role in the development of diabetic complications. Recent studies have shown that diabetes is associated with not only abnormal glucose metabolism but also abnormal ribose and fructose metabolism, although glucose is present at the highest concentration in humans. The glycation ability and contribution of ribose and fructose to diabetic complications remain unclear. Here, the glycation ability of ribose, fructose and glucose under a mimic physiological condition, in which the concentration of ribose or fructose was one-fiftieth that of glucose, was compared. Bovine serum albumin (BSA) was used as the working protein in our experiments. Ribose generated more AGEs and was markedly more cytotoxic to SH-SY5Y cells than fructose. The first-order rate constant of ribose glycation was found to be significantly greater than that of fructose glycation. LC-MS/MS analysis revealed 41 ribose-glycated Lys residues and 12 fructose-glycated residues. Except for the shared Lys residues, ribose reacted selectively with 17 Lys, while no selective Lys was found in fructose-glycated BSA. Protein conformational changes suggested that ribose glycation may induce BSA into amyloid-like monomers compared with fructose glycation. The levels of serum ribose were correlated positively with glycated serum protein (GSP) and diabetic duration in type 2 diabetes mellitus (T2DM), respectively. These results indicate that ribose has a greater glycation ability than fructose, while ribose largely contributes to the production of AGEs and provides a new insight to understand in the occurrence and development of diabetes complications.     

Generation and characterization of monoclonal antibody against Advanced Glycation End Products in chronic kidney disease

Advanced Glycation End Products (AGEs) are toxins that are involved in structural and functional alterations of several organs and tissues, resulting in various pathologies. Several types of AGEs have been described but carboxymethyllysine (CML) is the major antigenic AGE compound. In this study, three different immunogenic carrier proteins (KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; and HSA, human serum albumin) were modified by glycation. The glycated molecules were used to produce epitope-specific monoclonal antibodies able to recognize the CML domain and to detect uremic toxins in the serum of patients with chronic kidney disease (CKD). A competitive ELISA was standardized in order to quantify CML in the sera of CKD patients. An increase in uremic toxins can compromise the clinical condition of these patients, thus, the detection and quantification of these toxins should contribute to a better management and understanding of this disease.     

Glycation of antibodies: Modification,methods and potential effects on biological functions

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.