Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

MicroRNA-125b (miR-125b) has been implicated in a variety of diseases as either repressors or promoters, and plays crucial roles in many cellular processes such as cell differentiation, proliferation and apoptosis. Age-related cataract has become one of the most serious problems facing the aging population in the world. The purpose of this study was to investigate the role of miR-125b in the development of age-related cataract. We demonstrated that miR-125b was downregulated in both age-related cataract tissue and lens epithelial cell apoptosis induced by UV irradiation. We also identified the impact of miR-125b on apoptosis in a lens epithelial cell line. In vitro, miR-125b regulates human lens epithelial cell apoptosis at least in part by directly targeting p53. In addition, an inverse relationship between miR-125b and p53 expression was seen in age-related cataract tissue. In conclusion, this study suggests that miR-125b might be closely involved in the pathogenesis of cataract, and has the potential to be a diagnostic biomarker or even a therapeutic modality for cataract.

Hernia is a disease with defects in collagen synthesis/metabolism. However, the underlying mechanisms for hernia formation have not been fully defined. Tamoxifen is a selective estrogen receptor modulator and used for patients with breast cancer. Tamoxifen also has pleiotropic and side effects. Herein, we report that tamoxifen treatment resulted in an appearance of a large bulge in the low abdomen between the hind legs in male but not in female mice. The autopsy demonstrated that the low abdominal wall was broken and a large amount of intestine herniated out of the abdominal cavity. Histological analysis indicated that tamoxifen caused structural abnormalities in the low abdominal wall which was associated with decreased type II collagen content. Furthermore, we determined increased matrix metalloproteinase-2 (MMP-2) and MMP-13 expression in the tissue. In vitro, tamoxifen induced MMP-2 and MMP-13 expression in fibroblasts. The promoter activity analysis and ChIP assay demonstrate that induction of MMP-13 expression was associated with activation of JNK-AP-1 and ERK1/2 signaling pathways while induction of MMP-2 expression was related to activation of ERK1/2 signaling pathway. Taken together, our study establishes a novel murine hernia model, defines a severe side effect of tamoxifen, and suggests a caution to male patients receiving tamoxifen treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.

Fibroblast growth factor 21 (FGF21) is a modulator of energy homeostasis and is increased in human nonalcoholic liver disease (NAFLD) and after feeding of methionine- and choline-deficient diet (MCD), a conventional inducer of murine nonalcoholic steatohepatitis (NASH). However, the significance of FGF21 induction in the occurrence of MCD-induced NASH remains undetermined. C57BL/6J Fgf21-null and wild-type mice were treated with MCD for 1week. Hepatic Fgf21 mRNA was increased early after commencing MCD treatment independent of peroxisome proliferator-activated receptor (PPAR) α and farnesoid X receptor. While no significant differences in white adipose lipolysis were seen in both genotypes, hepatic triglyceride (TG) contents were increased in Fgf21-null mice, likely due to the up-regulation of genes encoding CD36 and phosphatidic acid phosphatase 2a/2c, involved in fatty acid (FA) uptake and diacylglycerol synthesis, respectively, and suppression of increased mRNAs encoding carnitine palmitoyl-CoA transferase 1α, PPARγ coactivator 1α, and adipose TG lipase, which are associated with lipid clearance in the liver. The MCD-treated Fgf21-null mice showed increased hepatic endoplasmic reticulum (ER) stress. Exposure of primary hepatocytes to palmitic acid elevated the mRNA levels encoding DNA damage-inducible transcript 3, an indicator of ER stress, and FGF21 in a PPARα-independent manner, suggesting that lipid-induced ER stress can enhance hepatic FGF21 expression. Collectively, FGF21 is induced in the early stage of MCD-induced NASH likely to minimize hepatic lipid accumulation and ensuing ER stress. These results provide a possible mechanism on how FGF21 is increased in NAFLD/NASH. Copyright © 2015. Published by Elsevier B.V.

Diabetic patients have increased likelihood of developing breast cancer. Advanced glycation endproducts (AGEs) underlie the pathogenesis of diabetic complications but their impact on breast cancer cells is not understood. This study aims to determine the effects of methylglyoxal-derived bovine serum albumin AGEs (MG-BSA-AGEs) on the invasive MDA-MB-231 breast cancer cell line. By performing cell counting, using wound-healing assay, invasion assay and zymography analysis, we found that MG-BSA-AGEs increased MDA-MB-231 cell proliferation, migration and invasion through Matrigel(TM) associated with an enhancement of matrix metalloproteinase (MMP)-9 activities, in a dose-dependent manner. Using Western blot and flow cytometry analyses, we demonstrated that MG-BSA-AGEs increased expression of the receptor for AGEs (RAGE) and phosphorylation of key signalling protein extracellular signal-regulated kinase (ERK)-1/2. Furthermore, in MG-BSA-AGE-treated cells, phospho-protein micro-array analysis revealed enhancement of phosphorylation of the ribosomal protein 70 serine S6 kinase beta 1 (p70S6K1), which is known to be involved in protein synthesis, the signal transducer and activator of transcription (STAT)-3 and the mitogen-activated protein kinase (MAPK) p38, which are involved in cell survival. Blockade of MG-BSA-AGE/RAGE interactions using a neutralizing anti-RAGE antibody inhibited MG-BSA-AGE-induced MDA-MB-231 cell processes, including the activation of signalling pathways. Throughout the study, non-modified BSA had a negligible effect. In conclusion, AGEs might contribute to breast cancer development and progression partially through the regulation of MMP-9 activity and RAGE signal activation. The up-regulation of RAGE and the concomitant increased phosphorylation of p70S6K1 induced by AGEs may represent promising targets for drug therapy to treat diabetic patients with breast cancer. Copyright © 2014. Published by Elsevier B.V.

We investigated the possibility that a cholesterol-rich milieu can accelerate response to pathogen-associated molecular patterns in order to elucidate mechanisms underlying aggravation of atherosclerosis after bacterial infection. The consumption of a high-cholesterol diet resulted in enhanced the expression of CD14 in arteries of ApoE(-/-) mice. 27-Hydroxycholesterol (27OHChol), the most abundant cholesterol oxide in atherosclerotic lesions, induced the significant expression of CD14 by THP-1 monocytic cells, but not by vascular smooth muscle cells or Jurkat T cells. Additions of lipopolysaccharide (LPS) to 27OHChol-treated THP-1 monocytic cells resulted in superinduction in terms of the gene transcription of CCL2 and the secretion of its gene product. In contrast, cholesterol did not cause increased the expression of CD14 in the aforementioned cells, and the addition of LPS to cholesterol-treated monocytic cells did not result in enhanced the expression of CCL2. The conditioned medium isolated from THP-1 cells exposed to 27OHChol plus LPS further induced the migration of monocytic cells in comparison with conditioned media obtained from THP-1 cells treated with 27OHChol or LPS alone. Treatment with 27OHChol also resulted in the enhanced secretion of MMP-9 and soluble CD14 (sCD14), and the secretion of sCD14 was blocked by a selective MMP-9 inhibitor. The inhibition of the ERK pathway resulted in significantly attenuated the secretion of sCD14 via mechanisms that were distinct from those by PI3K inhibition. We propose that 27OHChol can prime monocytes/macrophages by up-regulation of CD14 such that LPS-mediated inflammatory reaction is accelerated, thereby contributing to aggravated development of atherosclerotic lesions by enhancing recruitment of monocytic cells after infection with Gram-negative bacteria. Copyright © 2014. Published by Elsevier B.V.

Mutations in the liver isoform of the Phosphorylase Kinase (PhK) alpha subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK alpha subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK alpha functions and provide a framework for further experimental investigation.

Muscle wasting impairs physical performance, increases mortality and reduces medical intervention efficacy in chronic diseases and cancer. Developing proficient intervention strategies requires improved understanding of the molecular mechanisms governing muscle mass wasting and recovery. Involvement of muscle protein- and myonuclear turnover during recovery from muscle atrophy has received limited attention. The insulin-like growth factor (IGF)-I signaling pathway has been implicated in muscle mass regulation. As glycogen synthase kinase 3 (GSK-3) is inhibited by IGF-I signaling, we hypothesized that muscle-specific GSK-3β deletion facilitates the recovery of disuse-atrophied skeletal muscle. Wild-type mice and mice lacking muscle GSK-3β (MGSK-3β KO) were subjected to a hindlimb suspension model of reversible disuse-induced muscle atrophy and followed during recovery. Indices of muscle mass, protein synthesis and proteolysis, and post-natal myogenesis which contribute to myonuclear accretion, were monitored during the reloading of atrophied muscle. Early muscle mass recovery occurred more rapidly in MGSK-3β KO muscle. Reloading-associated changes in muscle protein turnover were not affected by GSK-3β ablation. However, coherent effects were observed in the extent and kinetics of satellite cell activation, proliferation and myogenic differentiation observed during reloading, suggestive of increased myonuclear accretion in regenerating skeletal muscle lacking GSK-3β. This study demonstrates that muscle mass recovery and post-natal myogenesis from disuse-atrophy are accelerated in the absence of GSK-3β. Copyright © 2014. Published by Elsevier B.V.

Sirt3, a mitochondrial NAD(+)-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F(o)F(1)ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F(o)F(1)ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F(o)F(1)ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F(o)F(1)ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F(o)F(1)ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ≅85% of mtDNA with 4977bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F(o)F(1)ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5-10μM of menadione impaired the Sirt3-mediated deacetylation and activation on F(o)F(1)ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F(o)F(1)ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.

Traumatic brain injury (TBI) is followed by a state of metabolic dysfunction, affecting the ability of neurons to use energy and support brain plasticity; there is no effective therapy to counteract the TBI pathology. Brain-derived neurotrophic factor (BDNF) has an exceptional capacity to support metabolism and plasticity, which highly contrasts with its poor pharmacological profile. We evaluated the action of a flavonoid derivative 7,8-dihydroxyflavone (7,8-DHF), a BDNF receptor (TrkB) agonist with the pharmacological profile congruent for potential human therapies. Treatment with 7,8-DHF (5mg/kg, ip, daily for 7days) was effective to ameliorate the effects of TBI on plasticity markers (CREB phosphorylation, GAP-43 and syntaxin-3 levels) and memory function in Barnes maze test. Treatment with 7,8-DHF restored the decrease in protein and phenotypic expression of TrkB phosphorylation after TBI. In turn, intrahippocampal injections of K252a, a TrkB antagonist, counteracted the 7,8-DHF induced TrkB signaling activation and memory improvement in TBI, suggesting the pivotal role of TrkB signaling in cognitive performance after brain injury. A potential action of 7,8-DHF on cell energy homeostasis was corroborated by the normalization in levels of PGC-1α, TFAM, COII, AMPK and SIRT1 in animals subjected to TBI. Results suggest a potential mechanism by which 7,8-DHF counteracts TBI pathology via activation of the TrkB receptor and engaging the interplay between cell energy management and synaptic plasticity. Since metabolic dysfunction is an important risk factor for the development of neurological and psychiatric disorders, these results set a precedent for the therapeutic use of 7,8-DHF in a larger context. Copyright © 2015. Published by Elsevier B.V.

We recently reported that deletion of the stress-regulated nuclear protein 1 (Nupr1) protected against obesity-associated metabolic alterations due to increased beta cell mass, but complete Nupr1 ablation was not advantageous since it led to insulin resistance on a normal diet. The current study used Nupr1 haplodeficient mice to investigate whether a partial reduction in Nupr1 expression conferred beneficial effects on glucose homeostasis. Islet number, morphology and area, assessed by immunofluorescence and morphometric analyses, were not altered in Nupr1 haplodeficient mice under normal diet conditions and nor was beta cell BrdU incorporation. Glucose and insulin tolerance tests indicated that there were no significant changes in in vivo insulin secretion and glucose clearance in Nupr1 haplodeficient mice, and beta cell function in vitro was normal. However, reduced Nupr1 expression decreased visceral fat deposition and significantly increased insulin sensitivity in vivo. In contrast to wild type animals, high fat diet-fed Nupr1 haplodeficient mice were not hyperinsulinaemic or glucose intolerant, and their sustained insulin sensitivity was demonstrated by appropriate insulin-induced Akt phosphorylation, as determined by Western blotting. At the molecular level, measurements of gene expression levels and promoter activities identified Nupr1-dependent inhibition of heat shock factor-1-induced heat shock protein 70 (Hsp70) expression as a mechanism through which Nupr1 regulates insulin sensitivity. We have shown for the first time that Nupr1 plays a central role in inhibiting Hsp70 expression in tissues regulating glucose homeostasis, and reductions in Nupr1 expression could be used to protect against the metabolic defects associated with obesity-induced insulin resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

Usher syndrome (USH), clinically and genetically heterogeneous, is the leading genetic cause of combined hearing and vision loss. USH is classified into three types, based on the hearing and vestibular symptoms observed in patients. Sixteen loci have been reported to be involved in the occurrence of USH and atypical USH. Among them, twelve have been identified as causative genes and one as a modifier gene. Studies on the proteins encoded by these USH genes suggest that USH proteins interact among one another and function in multiprotein complexes in vivo. Although their exact functions remain enigmatic in the retina, USH proteins are required for the development, maintenance and function of hair bundles, which are the primary mechanosensitive structure of inner ear hair cells. Despite the unavailability of a cure, progress has been made to develop effective treatments for this disease. In this review, we focus on the most recent discoveries in the field with an emphasis on USH genes, protein complexes and functions in various tissues as well as progress toward therapeutic development for USH. Copyright © 2014. Published by Elsevier B.V.

The aim was to determine the intestinal Ca(2+) absorption in type I diabetic rats after different times of STZ induction, as well as the gene and protein expression of molecules involved in both the transcellular and paracelluar Ca(2+) pathways. The redox state and the antioxidant enzymes of the enterocytes were also evaluated in duodenum from either diabetic or insulin-treated diabetic rats as compared to control rats. Male Wistar rats (150-200g) were divided into two groups: 1) controls and 2) STZ-induced diabetic rats (60mg/kg b.w.). A group of diabetic rats received insulin for five days. The insulin was adjusted daily to maintain a normal blood glucose level. Five 5 d after STZ injection, there was a reduction in the intestinal Ca(2+) absorption, which was maintained for 30 d and disappeared at 60 d. Similar changes occurred in the GSH and (˙)O2(-) levels. The protein expression of molecules involved in the transcellular pathway increased at 5 and 30 d returning to control values at 60 d. Their mRNA levels declined considerably at 60 d. The gene and protein expression of claudin 2 was upregulated at 30 d. Catalase activity increased at 5 and 30 d normalizing at 60 d. To conclude, type I D.m. inhibits the intestinal Ca(2+) absorption, which is transient leading to a time dependent adaptation and returning the absorptive process to normal values. The inhibition is accompanied by oxidative stress. When insulin is administered, the duodenal redox state returns to control values and the intestinal Ca(2+) absorption normalizes. Copyright © 2014. Published by Elsevier B.V.

Accumulation of misfolded forms of microtubule associated, neuronal protein tau causes neurofibrillary degeneration typical of Alzheimer's disease and other tauopathies. This process is accompanied by elevated cellular stress and concomitant deregulation of heat-shock proteins. We used a transgenic rat model of tauopathy to study involvement of heat shock protein 27 (Hsp27) in the process of neurofibrillary degeneration, its cell type specific expression and correlation with the amount of insoluble tau protein aggregates. The expression of Hsp27-mRNA is more than doubled and levels of Hsp27 protein tripled in aged transgenic animals with tau pathology. The data revealed a strong positive and highly significant correlation between Hsp27-mRNA and amount of sarkosyl insoluble tau. Interestingly, intracellular accumulation of insoluble misfolded tau protein in neurons was associated with overexpression of Hsp27 almost exclusively in reactive astrocytes, not in neurons. The topological dissociation of neuronally expressed pathological tau and the induction of astrocytic Hsp27, GFAP, and Vimentin along with up-regulation of microglia specific markers such as CD18, CD68 and C3 point to cooperation of astrocytes, microglia and neurons in response to intra-neuronal accumulation of insoluble tau. Our data suggest that over expression of Hsp27 represents a part of microglia-mediated astrocytic response mechanism in the process of neurofibrillary degeneration, which is not necessarily associated with neuroprotection and which in contrary may accelerate neurodegeneration in late stage of the disease. This phenomenon should be considered during development of disease modifying strategies for treatment of tauopathies and AD via regulation of activity of Hsp27. Copyright © 2015. Published by Elsevier B.V.

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remains to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation was also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Osteoporosis is a major public health issue that is expected to rise as the global population ages. Resveratrol (RES) is a plant polyphenol with various anti-aging properties. RES treatment of bone cells results in protective effects, but dose translation from in vitro studies to clinically relevant doses is limited since bioavailability is not taken into account. The aims of this review is to evaluate in vivo evidence for a role of RES supplementation in promoting bone health to reduced osteoporosis risk and potential mechanisms of action. Due to multiple actions on both osteoblasts and osteoclasts, RES has potential to attenuate bone loss resulting from different etiologies and pathologies. Several animal models have investigated the bone protective effects of RES supplementation. Ovariectomized rodent models of rapid bone loss due to estrogen-deficiency reported that RES supplementation improved bone mass and trabecular bone without stimulating other estrogen-sensitive tissues. RES supplementation prior to age-related bone loss was beneficial. The hindlimb unloaded rat model used to investigate bone loss due to mechanical unloading showed RES supplementation attenuated bone loss in old rats, but had inconsistent bone effects in mature rats. In growing rodents, RES increased longitudinal bone growth, but had no other effects on bone. In the absence of human clinical trials, evidence for a role of RES on bone heath relies on evidence generated by animal studies. A better understanding of efficacy, safety, and molecular mechanisms of RES on bone will contribute to the determination of dietary recommendations and therapies to reduce osteoporosis. This article is part of a Special Issue entitled: Resveratol: Challenges in translating pre-clincial findigns to iproved patient outcomes.

Emerging evidences suggest that chronic inflammation is one of the major causes of tumorigenesis. The role of inflammation in regulation of breast cancer progression is not well established. Recently Mediator of IRF3 Activation (MITA) protein has been identified that regulates NF-κB and IFN pathways. Role of MITA in the context of inflammation and cancer progression has not been investigated. In the current report, we studied the role of MITA in the regulation of cross talk between cell death and inflammation in breast cancer cells. The expression of MITA was significantly lower on in estrogen receptor (ER) positive breast cancer cells than ER negative cells. Similarly, it was significantly down regulated in tumor tissue as compared to the normal tissue. The overexpression of MITA in MCF-7 and T47D decreases the cell proliferation and increases the cell death by activation of caspases. MITA positively regulates NF-κB transcription factor, which is essential for MITA induced cell death. The activation of NF-κB induces TNF-α production which further sensitizes MITA induced cell death by activation of death receptor pathway through capsase-8. MITA expression decreases the colony forming units and migration ability of MCF-7 cells. Thus, our finding suggests that MITA act as a tumor suppressor which is down regulated during tumorigenesis providing survival advantage to tumor cell.

The tryptophan/kynurenine pathway (TKP) is the main route of tryptophan degradation and generates several neuroactive and immunomodulatory metabolites. Experimental and clinical data have clearly established that besides fat, muscle and liver, pancreatic islet tissue itself is a site of inflammation during obesity and type 2 diabetes. Therefore it is conceivable that pancreatic islet exposure to increased levels of cytokines may induce upregulation of islet kynurenine metabolism in a way resembling that seen in the brain in many neurodegenerative disorders. Using normal rat islets and the INS-1 β-cell line, we have demonstrated for the first time that: 1/only some TKP genes are constitutively expressed, both in β-cells as well as non β-cells; 2/ the regulatory enzyme indoleamine 2,3-dioxygenase (IDO1) is not constitutively expressed; 3/ IDO1 and kynurenine 3-monoxygenase (KMO) expression are potently activated by proinflammatory cytokines (IFN-γ, IL-1β) and glucolipotoxicity respectively, rather in β-cells than in non β-cells; 4/ Islet kynurenine/kynurenic acid production ratio is enhanced following IFN-γ and glucolipotoxicity; 5/ acute exposure to KYN potentiates glucose-induced insulin secretion by normal islets; 6/ oxidative stress or glucocorticoid modulates TKP genes only marginally. Pancreatic islets may represent a new target tissue for inflammation and glucolipotoxicity to activate the TKP. Since inflammation is now recognized as a crucial mechanism in the development of the metabolic syndrome and more specifically at the islet level, it is needed to evaluate the potential induction of the TKP in the endocrine pancreas during obesity and/or diabetes and its relationship to the islet cell functional alterations. Copyright © 2015. Published by Elsevier B.V.

NADPH oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30days. The agents were administered beginning at 1week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expression was increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. Apocynin treatment prevented increases in Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. Apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015. Published by Elsevier B.V.

We studied whether PPARβ/δ deficiency modifies the effects of high fructose intake (30% fructose in drinking water) on glucose tolerance and adipose tissue dysfunction, focusing on the CD36-dependent pathway that enhances adipose tissue inflammation and impairs insulin signaling. Fructose intake for 8weeks significantly increased body and liver weight, and hepatic triglyceride accumulation in PPARβ/δ-deficient mice but not in wild-type mice. Feeding PPARβ/δ-deficient mice with fructose exacerbated glucose intolerance and led to macrophage infiltration, inflammation enhanced mRNA and protein levels of CD36, and activation of the JNK pathway in white adipose tissue compared to those of water-fed PPARβ/δ-deficient mice. Cultured adipocytes exposed to fructose also exhibited increased CD36 protein levels and this increase was prevented by the PPARβ/δ activator GW501516. Interestingly, the levels of the nuclear factor E2-related factor 2 (Nrf2), a transcription factor reported to up-regulate Cd36 expression and to impair insulin signaling, were increased in fructose-exposed adipocytes whereas co-incubation with GW501516 abolished this increase. In agreement with Nrf2 playing a role in the fructose-induced CD36 protein level increases, the Nrf2 inhibitor trigonelline prevented the increase and the reduction in insulin-stimulated AKT phosphorylation caused by fructose in adipocytes. Protein levels of the well-known Nrf2 target gene NAD(P)H:quinone oxidoreductase 1 (Nqo1) were increased in water-fed PPARβ/δ-null mice, suggesting that PPARβ/δ deficiency increases Nrf2 activity; and this increase was exacerbated in fructose-fed PPARβ/δ-deficient mice. These findings indicate that the combination of high fructose intake and PPARβ/δ deficiency increases CD36 protein levels via Nrf2, a process that promotes chronic inflammation and insulin resistance in adipose tissue. Copyright © 2015. Published by Elsevier B.V.

A correlation between epilepsy and cellular redox imbalance has been suggested, although the mechanism by which oxidative stress (OS) can be implicated in this disorder is not clear. In the present study several oxidative stress markers and enzymes involved in OS have been determined. In particular, we examined the levels of 4-hydroxy-2-nonenal protein adducts (HNE-PA), a by-product of lipid peroxidation, and the activation of NADPH oxidase 2 (NOX2), as cellular source of superoxide (O2(-)), in surgically resected epileptic tissue from drug-resistant patients (N=50). In addition, we investigated whether oxidative-mediated protein damage can affect aquaporin-4 (AQP4), a water channel implicated in brain excitability and epilepsy. Results showed high levels of HNE-PA in epileptic hippocampus, in both neurons and glial cells and cytoplasmic positivity for p47(phox) and p67(phox) suggesting NOX2 activation. Interestingly, in epileptic tissue immunohistochemical localization of AQP4 was identified not only in perivascular astrocytic endfeet, but also in neurons. Nevertheless, negativity for AQP4 was observed in neurons in degeneration. Of note, HNE-mediated post-translational modifications of AQP4 were increased in epileptic tissues and double immunofluorescence clearly demonstrated co-localization of AQP4 and HNE-PA in epileptic hippocampal structures. The idea is that sudden, disorderly, and excessive neuronal discharges activates NOX2 with O2(-) production, leading to lipid peroxidation. The resulting generation of HNE targets AQP4, affecting water and ion balance. Therefore, we suggest that seizure induces oxidative damage as well as neuronal loss, thereby promoting neuronal hyperexcitability, also affecting water and ion balance by AQP4 modulation, and thus generating a vicious cycle. Copyright © 2014. Published by Elsevier B.V.

SUMOylation is a form of post-translational modification where small ubiquitin-like modifiers (SUMO) are covalently attached to target proteins to regulate their properties. SUMOylation has been demonstrated during cell stress and implicated in cellular stress response. However, it is largely unclear if SUMOylation contributes to the pathogenesis of kidney diseases, such as acute kidney injury (AKI). Here we have demonstrated a dynamic change of protein SUMOylation in ischemic and cisplatin nephrotoxic AKI in mice. In rat kidney proximal tubular cells (RPTC), cisplatin-induced SUMOylation was diminished by two antioxidants (N-acetylcysteine and dimethylurea), supporting a role of oxidative stress in the activation of SUMOylation. In addition, SUMOylation by SUMO-2/3, but not SUMO-1, was partially suppressed by pifithrin-alpha (a pharmacological inhibitor of p53), supporting a role of p53 in SUMOylation by SUMO-2/3. We further examined the role of SUMOylation during cisplatin treatment of RPTC cells by using ginkgolic acid (GA), a pharmacological inhibitor of SUMOylation. Pretreatment with GA suppressed SUMOylation and importantly, GA enhanced apoptosis during cisplatin incubation. Taken together, the results demonstrate the first evidence of SUMOylation in AKI and suggest that SUMOylation may play a cytoprotective role in kidney tubular cells. Copyright © 2014. Published by Elsevier B.V.

Renalischemia/reperfusion (I/R) injury is the most common cause of acutekidney injury, having a high rate of mortality and no effective therapy currently available. Apelin-13, a bioactive peptide, has been shown to inhibit the early lesions of diabetic nephropathy in several mouse models by us and others. To test whether apelin-13 protects against renal I/R induced injury, male rats were exposed to renal I/R injury with or without apelin-13 treatment for three days. Apelin-13 treatment markedly reduced the injury-induced tubular lesions, renal cell apoptosis, and normalized the injury induced renal dysfunction. Apelin-13 treatment inhibited the injury-induced elevation of inflammatory factors and Tgf-β1, as well as apoptosis. Apelin-13 treatment also inhibited the injury-induced elevation of histone methylation and Kmt2d, a histone methyltransferase of H3K4me2, following renal I/R injury. Furthermore, in cultured renal mesangial and tubular cells, apelin-13 suppressed the injury-induced elevation of Tgf-β1, apoptosis, H3K4me2 and Kmt2d under the in vitro hypoxia/reperfusion (H/R) conditions. Consistently, over-expression of apelin significantly inhibited H/R-induced elevation of TGF-β1, apoptosis, H3K4me2 and Kmt2d. The present study therefore suggests apelin-13 may be a therapeutic candidate for treating acutekidney injury. Copyright © 2015. Published by Elsevier B.V.

Olaparib (AZD-2281, Ku-0059436) is an orally bioavailable and well-tolerated poly(ADP-ribose) polymerase (PARP) inhibitor currently under investigation in patients with solid tumors. To study the clinical potential of olaparib as a single-agent for the treatment of Acute Myeloid Leukemia (AML) patients, we analyzed the in vitro sensitivity of AML cell lines and primary blasts. Clinically achievable concentrations of olaparib were able to induce cell death in the majority of primary AML case samples (88%) and tested cell lines. At these concentrations, olaparib preferentially killed leukemic blasts sparing normal lymphocytes derived from the same patient and did not substantially affect the viability of normal bone marrow and CD34-enriched peripheral blood cells obtained from healthy donors. Most primary AML analyzed were characterized by low BRCA1 mRNA level and undetectable protein expression that likely contributed to explain their sensitivity to olaparib. Noteworthy, while PARP1 over-expression was detected in blasts not responsive to olaparib, phosphorylation of the histone H2AFX (γH2AX) was associated with drug sensitivity. As to genetic features of tested cases the highest sensitivity was shown by a patient carrying a 11q23 deletion. The high sensitivity of AML blasts and the identification of biomarkers potentially able to predict response and/or resistance may foster further investigation of olaparib monotherapy for AML patients unfit to conventional chemotherapy. Copyright © 2014. Published by Elsevier B.V.

Dysfunction of complex I (CI) of the mitochondrial electron transport chain (ETC) features prominently in human pathology. Cell models of ETC dysfunction display adaptive survival responses that still are poorly understood but of relevance for therapy development. Here we comprehensively examined how primary human skin fibroblasts adapt to chronic CI inhibition. CI inhibition triggered transient and sustained changes in metabolism, redox homeostasis and mitochondrial (ultra)structure but no cell senescence/death. CI-inhibited cells consumed no oxygen and displayed minor mitochondrial depolarization, reverse-mode action of complex V, a slower proliferation rate and futile mitochondrial biogenesis. Adaptation was neither prevented by antioxidants nor associated with lower ATP or increased PGC1-α/SIRT1/mTOR levels. Survival of CI-inhibited cells was strictly glucose-dependent and accompanied by increased AMPK-α phosphorylation, which occurred without changes in ATP or cytosolic calcium levels. Conversely, cells devoid of AMPK-α died upon CI inhibition. Chronic CI inhibition did not increase mitochondrial superoxide levels or cellular lipid peroxidation and was paralleled by a specific increase in SOD2/GR, whereas SOD1/CAT/Gpx1/Gpx2/Gpx5 levels remained unchanged. Upon hormone stimulation, fully adapted cells displayed aberrant cytosolic and ER calcium handling due to hampered ATP fueling of ER calcium pumps. Using a cell model of CI inhibition we explored the temporal adaptation to CI dysfunction and circumvent problems associated with patient-derived cell systems. It is concluded that CI dysfunction triggers an adaptive program that depends on extracellular glucose and AMPK-α. This response avoids cell death by suppressing energy crisis, oxidative stress induction and substantial mitochondrial depolarization. Copyright © 2014. Published by Elsevier B.V.

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.

Long chain fatty acids bind to cartinine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca(2+) dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10μM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20μM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca(2+) leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca(2+) leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongrekic acid and NAC significantly reduced spontaneous Ca(2+) waves occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca(2+) handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions. Copyright © 2015. Published by Elsevier B.V.

Adiponectin (APN), an adipokine, exerts an anti-inflammatory and anti-cancerous activity with its role in glucose and lipid metabolism and its absence related to several obesity related malignancies including colorectal cancer. The aim of this study is to determine the effect of APN deficiency on the chronic inflammation-induced colon cancer. This was achieved by inducing inflammation and colon cancer in both APN knockout (KO) and C57B1/6 wild type (WT) mice. They were divided into four treatment groups (n=6): 1) control (no treatment); 2) treatment with three cycles of dextran sodium sulfate (DSS); 3) weekly doses of 1,2-dimethylhydrazine (DMH) (20mg/kg of mouse body weight) for twelve weeks; 4) a single dose of DMH followed by 3 cycles of DSS (DMH+DSS). Mice were observed for diarrhea, stool hemoccult, and weight loss and were sacrificed on day 153. Tumor area and number were counted. Colonic tissues were collected for Western blot and immunohistochemistry analyses. APNKO mice were more protected than WT mice from DSS induced colitis during first DSS cycle, but lost this protection during the second and the third DSS cycles. APNKO mice had significantly severe symptoms and showed greater number and larger area of tumors with higher immune cell infiltration and inflammation than WT mice. This result was further confirmed by proteomic study including pSTAT3, pAMPK and Cox-2 by western blot and Immunohistochemistry. Conclusively, APN deficiency contributes to inflammation-induced colon cancer. Hence, APN may play an important role in colorectal cancer prevention by modulating genes involved in chronic inflammation and tumorigenesis.

SLC25A13 (citrin or aspartate-glutamate carrier 2) is located in the mitochondrial membrane in the liver and its genetic deficiency causes adult-onset type II citrullinemia (CTLN2). CTLN2 is one of the urea cycle disorders characterized by sudden-onset hyperammonemia due to reduced argininosuccinate synthase activity. This disorder is frequently accompanied with hepatosteatosis in the absence of obesity and ethanol consumption. However, the precise mechanism of steatogenesis remains unclear. The expression of genes associated with fatty acid (FA) and triglyceride (TG) metabolism was examined using liver samples obtained from 16 CTLN2 patients and compared with 7 healthy individuals. Although expression of hepatic genes associated with lipogenesis and TG hydrolysis was not changed, the mRNAs encoding enzymes/proteins involved in FA oxidation (carnitine palmitoyl-CoA transferase 1α, medium- and very-long-chain acyl-CoA dehydrogenases, and acyl-CoA oxidase 1), very-low-density lipoprotein secretion (microsomal TG transfer protein), and FA transport (CD36 and FA-binding protein 1), were markedly suppressed in CTLN2 patients. Serum concentrations of ketone bodies were also decreased in these patients, suggesting reduced mitochondrial β-oxidation activity. Consistent with these findings, the expression of peroxisome proliferator-activated receptor α (PPARα), a master regulator of hepatic lipid metabolism, was significantly down-regulated. Hepatic PPARα expression was inversely correlated with severity of steatosis and circulating ammonia and citrulline levels. Additionally, phosphorylation of c-Jun-N-terminal kinase was enhanced in CTLN2 livers, which was likely associated with lower hepatic PPARα. Collectively, down-regulation of PPARα is associated with steatogenesis in CTLN2 patients. These findings provide a novel link between urea cycle disorder, lipid metabolism, and PPARα. (248 words). Copyright © 2014. Published by Elsevier B.V.

Diabetic neuronal damage results from hyperglycemia followed by increased formation of advanced glycosylation end products (AGEs), which lead to neurodegeneration, although the molecular mechanisms are still not well understood. Metformin, one of the most widely used anti-diabetic drugs, exerts its effects in part by activation of AMP-activated protein kinase (AMPK). AMPK is a critical evolutionarily conserved enzyme expressed in the liver, skeletal muscle and brain, and promotes cellular energy homeostasis and biogenesis by regulating several metabolic processes. While the mechanisms of AMPK as a metabolic regulator are well established, the neuronal role for AMPK is still unknown. In the present study, human neural stem cells (hNSCs) exposed to AGEs had significantly reduced cell viability, which correlated with decreased AMPK and mitochondria associated gene/protein (PGC1α, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3 and 9 activity. Co-treatment with metformin significantly abrogated the AGEs-mediated effects in hNSCs. Metformin prevented AGEs induced cytochrome c release from mitochondria into cytosol in the hNSCs. Metformin also signficantly rescued hNSCs from AGEs-mediated mitochondrial deficiency (lower ATP, D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Furthermore, co-treatment of hNSCs with metformin significantly blocked AGE-mediated reductions in the expression levels of several neuroprotective genes (PPARγ, Bcl-2 and CREB). These findings extend our understanding of the molecular mechanisms of both AGE-induced neuronal toxicity, and AMPK-dependent neuroprotection by metformin. This study further suggests AMPK may be a potential therapeutic target for treating diabetic neurodegeneration. Copyright © 2015. Published by Elsevier B.V.

Ageing of the neuromuscular system in elderhood ingravescently contributes to slowness, weakness, falling and death, often accompanied by numbness and pain. This article is to put in perspective examples from a half-century of personal and team neuromuscular histochemical-pathological and clinical-pathological research, including a number of lucky and instructive accomplishments identifying new treatments and new diseases. A major focus currently is on some important, still enigmatic, aspects of the ageing neuromuscular system. It is also includes some of the newest references of others on various closely-related aspects of this ageing system. The article may help guide others in their molecular-based endeavors to identify paths leading to discovering new treatments and new pathogenic aspects. These are certainly needed - our ageing and unsteady constituents are steadily increasing. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis. Copyright © 2014. Published by Elsevier B.V.

Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca(2+) signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca(2+) chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion. Copyright © 2015. Published by Elsevier B.V.

Rapid remodeling of the actin cytoskeleton in the pre- and/or post-synaptic compartments is responsible for the regulation of neuronal plasticity, which is an important process for learning and memory. Cofilin1 plays an essential role in these processes and a dysregulation of its activity was associated with the cognitive decline observed during normal ageing and Alzheimer's disease (AD). To understand the mechanism(s) regulating Cofilin1 activity we evaluated changes occurring with regard to Cofilin1 and its up-stream regulators Lim kinase-1 (LIMK1) and Slingshot phosphatase-1 (SSH1) in (i) human AD brain, (ii) 1-, 4-, and 10-months old APP/PS1 mice, (iii) wild type 3-, 8-, 12-, 18- and 26-months old mice, as well as in cellular models including (iv) mouse primary cortical neurons (PCNs, cultured for 5, 10, 15 and 20days in vitro) and (v) mouse embryonic fibroblasts (MEF). Interestingly, we found an increased Cofilin1 phosphorylation/inactivation with age and AD pathology, both in vivo and in vitro. These changes were associated with a major inactivation of SSH1. Interestingly, inhibition of γ-secretase activity with Compound-E (10μM) prevented Cofilin1 phosphorylation/inactivation through an increase of SSH1 activity in PCNs. Similarly, MEF cells double knock-out for γ-secretase catalytic subunits presenilin-1 and -2 (MEFDKO) showed a strong decrease of both Cofilin1 and SHH1 phosphorylation, which were rescued by the overexpression of human γ-secretase. Together, these results shed new light in understanding the molecular mechanisms promoting Cofilin1 dysregulation, both during ageing and AD. They further have the potential to impact the development of therapies to safely treat AD.

Alzheimer's disease (AD) is a devastating disorder that strikes 1 in 10 Americans over the age of 65, and almost half of all Americans over 85years old. The odds of an individual developing Alzheimer's disease double every five years after the age of 65. While it has become increasingly common to meet heart attack or cancer survivors, there are no Alzheimer's disease survivors. There is mounting evidence that dietary polyphenols, including resveratrol, may beneficially influence Alzheimer's disease (AD). Based on this consideration, several studies reported in the last few years were designed to validate sensitive and reliable translational tools to mechanistically characterize brain bioavailable polyphenols as disease-modifying agents to help prevent the onset of AD dementia and other neurodegenerative disorders. Several research groups worldwide with expertise in AD, plant biology, nutritional sciences, and botanical sciences have reported very high quality studies that ultimately provided the necessary information showing that polyphenols and their metabolites, which come from several dietary sources, including grapes, cocoa etc., are capable of preventing AD. The ultimate goal of these studies was to provide novel strategies to prevent the disease even before the onset of clinical symptoms. The studies discussed in this review article provide support that the information gathered in the last few years of research will have a major impact on AD prevention by providing vital knowledge on the protective roles of polyphenols, including resveratrol.

Our previous studies have shown that the 3' end of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in colorectal cancer (CRC) cell proliferation and migration/invasion in vitro. The role and mechanism of MALAT1 in CRC metastasis in vivo, however, remain largely unknown. In the present study, we found that MALAT1 was up-regulated in human primary CRC tissues with lymph node metastasis. Overexpression of MALAT1 via RNA activation promoted CRC cell proliferation, invasion and migration in vitro, and stimulated tumor growth and metastasis in mice in vivo. Conversely, knockdown of MALAT1 inhibited CRC tumor growth and metastasis. MALAT1 regulated at least 243 genes in CRC cells in a genome-wide expression profiling. Among these genes, PRKA kinase anchor protein 9 (AKAP-9) was significantly up-regulated at both mRNA and protein levels. AKAP-9 was highly expressed in CRC cells with metastatic potential and human primary CRC tissues with lymph node metastasis, but not in normal cells or tissues. Importantly, knockdown of AKAP-9 blocked MALAT1-mediated CRC cell proliferation, migration and invasion. These data indicate that MALAT1 may promote CRC tumor development via its target protein AKAP-9. Copyright © 2014. Published by Elsevier B.V.

CEACAM6 is a member of glycosylphosphatidylinositol (GPI)-linked immunoglobulin superfamily that is implicated in a variety of human cancers. In our previous study, we reported that CEACAM6 was overexpressed in gastric cancer (GC) tissues and promoted cancer metastasis. The purpose of this study is to determine the role of CEACAM6 in tumor angiogenesis and vasculogenic mimicry (VM) formation. We found that overexpressed CEACAM6 promoted tubule formation dependent on HUVEC cells and VM formation of GC cells; opposing results were achieved in CEACAM6-silenced groups. Moreover, we found that mosaic vessels formed by HUVEC cells and GC cells were observed in vitro by 3D-culture assay. Overexpressed CEACAM6 in GC cells promoted tumor growth, VEGF expression and VM structures formation in vivo. In accordance with these observations, we found that phosphorylation of FAK and phosphorylation of paxillin were up-regulated in CEACAM6-overexpressing GC cells, and FAK inhibitor Y15 could reduce tubule and VM formation. These findings suggest that CEACAM6 promotes tumor angiogenesis and VM formation may via FAK signaling in GC and CEACAM6 maybe a new target for cancer anti-vascular treatment. Copyright © 2015. Published by Elsevier B.V.

Vascular endothelial cells (ECs) are responsible for post-ischemic angiogenesis, a process that is regulated by reactive oxygen species. Recent studies indicate that endothelial Nox4 based NADPH oxidase may have a key role. This study examines the role of endothelial Nox4 in ischemia-induced angiogenesis and explores the potential mechanisms involved. Mouse lines overexpressing human Nox4 wild type (EWT) or its dominant negative form P437H (EDN) specifically in the endothelium were used. Non-transgenic littermate mice (NTg) were used as controls. Following hind limb ischemia, blood flow recovery was enhanced in EWT and was impaired in EDN compared with NTg. The critical angiogenesis regulating genes vascular endothelial growth factor receptor 2 (VEGFR2), endothelial nitric oxide synthase (eNOS) and transforming growth factor β1 (TGFβ1) were upregulated in EWT both in the ischemic muscle and in heart ECs, while TGFβ1 was downregulated in EDN ECs. In EC, both VEGFA and TGFβ1 stimulated EC proliferation, migration, and capillary-like network formation in EWT but failed to do so in EDN. Application of TGFβ1 increased both VEGFR2 and eNOS expression levels, whereas blocking TGFβ1 or addition of catalase inhibited the phosphorylation of VEGFR2 and eNOS, indicating H2O2 and TGFβ1 signaling downstream of Nox4 is critical to maintain EC angiogenic functions. Use of cell specific transgenic mice with both upregulation and downregulation of endothelial Nox4 indicate several mechanisms linked to Nox4 play a role in angiogenesis. Endothelial Nox4 regulates ischemia-induced angiogenesis, likely through H2O2- and TGFβ1-mediated activation of cell signaling pathways essential for endothelial function.

Diabetes mellitus is a serious disease affecting about 5% of people worldwide. Diabetes is characterized by hyperglycemia and impairment in insulin secretion and/or action. Moreover, diabetes is associated with metabolic abnormalities and serious complications. Resveratrol is a natural, biologically active polyphenol present in different plant species and known to have numerous health-promoting effects in both animals and humans. Anti-diabetic action of resveratrol has been extensively studied in animal models and in diabetic humans. In animals with experimental diabetes, resveratrol has been demonstrated to induce beneficial effects that ameliorate diabetes. Resveratrol, among others, improves glucose homeostasis, decreases insulin resistance, protects pancreatic β-cells, improves insulin secretion and ameliorates metabolic disorders. Effects induced by resveratrol are strongly related to the capability of this compound to increase expression/activity of AMPK and SIRT1 in various tissues of diabetic subjects. Moreover, anti-oxidant and anti-inflammatory effects of resveratrol were shown to be also involved in its action in diabetic animals. Preliminary clinical trials show that resveratrol is also effective in type 2 diabetic patients. Resveratrol may, among others, improve glycemic control and decrease insulin resistance. These results show that resveratrol holds great potential to treat diabetes and would be useful to support conventional therapy. This article is part of a Special Issue entitled: Resveratol: Challenges in translating pre-clincial findigns to iproved patient outcomes, guest edited by J. Dyck and P. Schrauwen. Copyright © 2014 Elsevier B.V. All rights reserved.

Recently, Aβ peptide variants with an N-terminal truncation and pyroglutamate modification were identified and shown to be highly neurotoxic and prone to aggregation. This modification of Aβ is catalyzed by glutaminyl cyclase (QC) and pharmacological inhibition of QC diminishes Aβ deposition and accompanying gliosis and ameliorates memory impairment in transgenic mouse models of Alzheimer's disease (AD). QC expression was initially described in the hypothalamus, where thyrotropin-releasing hormone (TRH) is one of its physiological substrates. In addition to its hormonal role, a novel neuroprotective function of TRH following excitotoxicity and Aβ-mediated neurotoxicity has been reported in the hippocampus. Functionally matching this finding, we recently demonstrated QC expression by hippocampal interneurons in mouse brain. Here, we detected neuronal co-expression of QC and TRH in the hippocampus of young adult wild type mice using double immunofluorescence labeling. This provides evidence for TRH being a physiological QC substrate in hippocampus. Additionally, in neocortex of aged but not of young mice transgenic for amyloid precursor protein an increase of QC mRNA levels was found compared to wild type littermates. This phenomenon was not observed in hippocampus, which is later affected by Aβ pathology. However, in hippocampus of transgenic - but not of wild type mice - a correlation between QC and TRH mRNA levels was revealed. This co-regulation of the enzyme QC and its substrate TRH was reflected by a co-induction of both proteins in reactive astrocytes in proximity of Aβ deposits. Also, in primary mouse astrocytes a co-induction of QC and TRH was demonstrated upon Aβ stimulation. Copyright © 2014 Elsevier B.V. All rights reserved.

Despite intensive research, the pathways that mediate calcium (Ca(2+))-stimulated glucose transport in striated muscle remain elusive. Since the sarcoplasmic reticulum calcium ATPase (SERCA) pump tightly regulates cytosolic [Ca(2+)], we investigated whether the SERCA pump is a major regulator of cardiac glucose transport. We used healthy and insulin-deficient diabetic transgenic (TG) mice expressing SERCA1a in the heart. Active cell surface glucose transporter (GLUT)-4 was measured by a biotinylated photolabeled assay in the intact perfused myocardium and isolated myocytes. In healthy TG mice, cardiac-specific SERCA1a expression increased active cell-surface GLUT4 and glucose uptake in the myocardium, as well as whole body glucose tolerance. Diabetes reduced active cell-surface GLUT4 content and glucose uptake in the heart of wild type mice, all of which were preserved in diabetic TG mice. Decreased basal AS160 and increased proportion of calmodulin-bound AS160 paralleled the increase in cell surface GLUT4 content in the heart of TG mice, suggesting that AS160 regulates GLUT trafficking by a Ca(2+)/calmodulin dependent pathway. In addition, cardiac-specific SERCA1a expression partially rescues hyperglycemia during diabetes. Collectively, these data suggested that the SERCA pump is a major regulator of cardiac glucose transport by an AS160 dependent mechanism during healthy and insulin-deficient state. Our data further indicated that cardiac-specific SERCA overexpression rescues diabetes induced-alterations in cardiac glucose transport and improves whole body glucose homeostasis. Therefore, findings from this study provide novel mechanistic insights linking upregulation of the SERCA pump in the heart as a potential therapeutic target to improve glucose metabolism during diabetes. Copyright © 2015. Published by Elsevier B.V.

Apart from control of circulating fluid, atrial natriuretic peptide (ANP) exhibits anti-inflammatory effects in the lung. However, molecular mechanisms of ANP anti-inflammatory effects are not well-understood. Peripheral microtubule (MT) dynamics is essential for agonist-induced regulation of vascular endothelial permeability. Here we studied the role of MT-dependent signaling in ANP protective effects against endothelial cell (EC) barrier dysfunction and acute lung injury induced by Staphylococcus aureus-derived peptidoglican-G (PepG). PepG-induced vascular endothelial dysfunction was accompanied by MT destabilization and disruption of MT network. ANP attenuated PepG-induced MT disassembly, NFkB signaling and activity of MT-associated Rho activator GEF-H1 leading to attenuation of EC inflammatory activation reflected by expression of adhesion molecules ICAM1 and VCAM1. ANP-induced EC barrier preservation and MT stabilization were linked to phosphorylation and inactivation of MT-depolymerizing protein stathmin. Expression of stathmin phosphorylation-deficient mutant abolished ANP protective effects against PepG-induced inflammation and EC permeability. In contrast, siRNA-mediated stathmin knockdown prevented PepG-induced peripheral MT disassembly and endothelial barrier dysfunction. ANP protective effects in a murine model of PepG-induced lung injury were associated with increased phosphorylation of stathmin, while exacerbated lung injury in the ANP knockout mice was accompanied by decreased pool of stable MT. Stathmin knockdown in vivo reversed exacerbation of lung injury in the ANP knockout mice. These results show a novel MT-mediated mechanism of endothelial barrier protection by ANP in pulmonary EC and animal model of PepG-induced lung injury via stathmin-dependent control of MT assembly. Copyright © 2014. Published by Elsevier B.V.

Primary open angle glaucoma (POAG) is a degenerative disease commonly associated with aging and elevated intraocular pressure (IOP). Higher resistance to aqueous humor (AH) outflow through the trabecular meshwork (TM) generates the elevated IOP in POAG; unfortunately the underlying molecular mechanisms responsible for elevated resistance are unknown. It is widely accepted, however, that differences between normal and POAG TM tissues are presumably a consequence of cellular dysfunction. Here, we investigated the autophagic function and response to chronic oxidative stress in TM cells isolated from glaucomatous and age-matched donor eyes. Glaucomatous TM cells showed elevated senescence-associated-beta-galactosidase (SA-β-Gal) and cellular lipofuscin, together with decreased steady-state levels of LC3B-II, decreased levels of pRPS6K-T389 and reduced proteolysis of long-live proteins. Moreover, the glaucomatous cultures failed to activate autophagy when exposed to hyperoxic conditions. These results strongly suggest mTOR-dependent dysregulation of the autophagic pathway in cells isolated from the glaucomatous TM. Such dysregulated autophagic capacity can have a detrimental impact in outflow pathway tissue, i.e mechanotransduction, and thus represent an important factor contributing to the progression of the disease. Copyright © 2014. Published by Elsevier B.V.

We have explored the mechanisms underlying ethanol-induced mitochondrial dynamics disruption and mitophagy. Ethanol increases mitochondrial fission in a concentration-dependent manner through Drp1 mitochondrial translocation and OPA1 proteolytic cleavage. ARPE-19 (a human retinal pigment epithelial cell line) cells challenged with ethanol showed mitochondrial potential disruptions mediated by alterations in mitochondrial complex IV protein level and increases in mitochondrial reactive oxygen species production. In addition, ethanol activated the canonical autophagic pathway, as denoted by autophagosome formation and autophagy regulator elements including Beclin1, ATG5-ATG12 and P-S6 kinase. Likewise, autophagy inhibition dramatically increased mitochondrial fission and cell death, whereas autophagy stimulation rendered the opposite results, placing autophagy as a cytoprotective response aimed to remove damaged mitochondria. Interestingly, although ethanol induced mitochondrial Bax translocation, this episode was associated to cell death rather than mitochondrial fission or autophagy responses. Thus, Bax required 600mM ethanol to migrate to mitochondria, a concentration that resulted in cell death. Furthermore, mouse embryonic fibroblasts lacking this protein respond to ethanol by undergoing mitochondrial fission and autophagy but not cytotoxicity. Finally, by using the specific mitochondrial-targeted scavenger MitoQ, we revealed mitochondria as the main source of reactive oxygen species that trigger autophagy activation. These findings suggest that cells respond to ethanol activating mitochondrial fission machinery by Drp1 and OPA1 rather than bax, in a manner that stimulates cytoprotective autophagy through mitochondrial ROS. Copyright © 2015. Published by Elsevier B.V.

The human BCL6 gene encodes a transcriptional repressor that is crucial for germinal center B cell development and T follicular helper cell differentiation. It is involved in the pathogenesis of certain human lymphomas. In an effort to identify targets of BCL6 repression, we used a previously described cell system in which BCL6 repressive effects are inhibited, followed by subtractive hybridization, and identified the integral membrane 2B gene (ITM2B, formerly BRI2) as a potential target. Here we show that BCL6 can bind to its preferential consensus binding site within the first intron of ITM2B and represses its transcription. Knockdown of endogenous BCL6 in a human B cell lymphoma line increases ITM2B expression. Further, there is an inverse relationship between the expression levels of BCL6 and ITM2B proteins in 16 human B and T cell lymphomas studied by immunohistochemistry. Both the BCL6 and ITM2B proteins are expressed ubiquitously. Similar to some other targets of BCL6, a short form of the ITM2B protein generated by alternative splicing induces apoptosis in hematopoietic cell lines. Molecular alterations in the ITM2B gene are associated with two neurodegenerative diseases, Familial British and Familial Danish dementia. ITM2B dysfunction also may be relevant for the development of Alzheimer's disease. Our data confirm ITM2B as a target of BCL6 repression in lymphoma. A further understanding of the genes that function as regulators of the ITM2B protein may provide insights for the development of new molecular tools not only for targeted lymphoma therapy but also for the treatment of these dementias. Copyright © 2014. Published by Elsevier B.V.

Hepatocellular carcinoma (HCC) is an invasive cancer with a high rate of recurrence and metastasis. Agents with anti-proliferative as well as anti-metastatic activity will be ideal for effective treatment. Here, we demonstrated that berberine, an isoquinoline alkaloid, harbored potent anti-metastatic and anti-proliferative activities in vivo. Using an orthotopic model of HCC (MHCC-97L), which spontaneously develops lung metastases (one of the most common sites of HCC metastasis), we found that berberine treatment (10mg/kg/2days) significantly reduced lung metastasis from the liver tumors by ~85% (quantitated by bioluminescence emitted from lung metastases). Histological examination also confirmed the reduced incidence and number of lung metastases in berberine-treated mice. Furthermore, berberine effectively suppressed extra-tumor invasion of the primary HCC implant into the surrounding normal liver tissue, illustrating its potent anti-metastatic action in vivo. Consistent with previous reports in other cancer, berberine's anti-tumor activity was accompanied by suppression of cellular proliferation, invasiveness and HIF-1α/VEGF signaling. Strikingly, further mechanistic investigation revealed that berberine exerted profound inhibitory effect on the expression of Id-1, which is a key regulator for HCC development and metastasis. Bereberine could suppress the transcription level of Id-1 through inhibiting its promotor activity. Specific downregulation of Id-1 by knocking down its RNA transcripts in HCC cells inhibited cellular growth, invasion and VEGF secretion, demonstrating the functional relevance of Id-1 downregulation induced by berberine. Lastly, berberine's anti-proliferative and anti-invasive activities could be partially rescued by Id-1 overexpression in HCC models, revealing a novel anti-cancer/anti-invasive mechanism of berberine via Id-1 suppression. Copyright © 2014. Published by Elsevier B.V.

Increased oxidative stress and cardiac inflammation have been implicated in the pathogenesis of diabetic cardiomyopathy (DCM). We previously found that a novel chalcone derivative, L6H9, was able to reduce LPS-induced inflammatory response in macrophages. This study was designed to investigate its protective effects on DCM and the underlying mechanisms. H9C2 cells were cultured with DMEM containing 33mmol/L of glucose in the presence or absence of L6H9. Pretreatment with L6H9 significantly reduced high glucose-induced inflammatory cytokine expression, ROS level increase, mitochondrial dysfunction, cell apoptosis, fibrosis, and hypertrophy in H9c2 cells, which may be mediated by NF-κB inhibition and Nrf2 activation. In mice with STZ-induced diabetes, oral administration of L6H9 at 20mg/kg/day for 8weeks significantly decreased the cardiac cytokine and ROS level, accompanied by decreasing cardiac apoptosis and hypertrophy, and, finally, improved histological abnormalities and fibrosis, without affecting the hyperglycemia. L6H9 also attenuated the diabetes-induced NF-κB activation and Nrf2 decrease in diabetic hearts. These results strongly suggest that L6H9 may have great therapeutic potential in the treatment of DCM via blockage of inflammation and oxidative stress. This study also provides a deeper understanding of the regulatory role of Nrf2 and NF-κB in DCM, indicating that they may be important therapeutic targets for diabetic complications. Copyright © 2015. Published by Elsevier B.V.

Drug resistance is the major cause of failure of cancer chemotherapy in ovarian cancer. However, the molecular mechanisms on the regulation of drug resistance are not fully understood. Here we showed that Trx1 and FOXO1 were involved in paclitaxel (PTX)-induced drug resistance in ovarian cancer A2780 cells. PTX induced reactive oxygen species (ROS) and resulted in Trx1 and FOXO1 nuclear translocation. We further found that Trx1 bound to FOXO1 and enhanced FOXO1 transcriptional activity, however Trx1 C69S mutant which is barely detected in the nucleus downregulated Trx1-FOXO1 interaction and Trx1-induced FOXO1 transcriptional activation. Silencing of FOXO1 abrogated Trx1-induced drug resistance. Trx1 increased FOXO1-induced drug resistance, while Trx1 C69S mutant completely abolished the regulation of FOXO1-mediated drug resistance by Trx1. These findings provided a novel mechanism on Trx1/FOXO1 signaling in drug resistance in ovarian cancer cells. Copyright © 2014. Published by Elsevier B.V.