Thioredoxin-interacting Protein Levels Research Articles

Aralia elata (Miq) Seem Extract Decreases O-GlcNAc Transferase Expression and Retinal Cell Death in Diabetic Mice.

Aralia elata (Miq) Seem (AES) is a medicinal plant used in traditional Chinese and Korean medicine for the treatment of several diseases, including diabetes. This study aimed to investigate the neuroprotective effect of AES extract against high glucose-induced retinal injury in diabetic mice. AES extract (20 and 100 mg/kg body weight) was orally administered to control mice or mice with streptozotocin-induced diabetes. Protein levels of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), carbohydrate-responsive element-binding protein (ChREBP), sterol regulatory element-binding protein (SREBP)-1, thioredoxin-interacting protein (TXNIP), fatty acid synthase (FAS), and acetyl CoA carboxylase (ACC) were analyzed by western blotting. Colocalization of terminal deoxynucleotide transferase-mediated dUTP nicked-end labeling (TUNEL)-positive ganglion cells and OGT, ChREBP, or TXNIP were monitored using double immunofluorescence analysis. Interaction between ChREBP and OGT was assessed using coimmunoprecipitation analysis. AES extract protected the retinas from neuronal injury and decreased levels of OGT, ChREBP, TXNIP, SREBP-1, FAS, and ACC in the diabetic retinas. AES extract reduced colocalization of TUNEL-positive ganglion cells and OGT, ChREBP, or TXNIP in the diabetic retinas. Coimmunoprecipitation analysis indicated that AES extract reduced interaction between ChREBP and OGT and attenuated ganglion cell death in diabetic retinas. Moreover, the ChREBP that colocalized with OGT or the TUNEL signal was significantly decreased in diabetic mice treated with AES extract. These findings show that AES extract can alleviate OGT-, ChREBP-, TXNIP-, or SREBP-1-related retinal injury in diabetic retinopathy.

A novel indication of thioredoxin-interacting protein as a tumor suppressor gene in malignant glioma.

Malignant glioma, the most common form of primary brain tumor, is associated with substantial morbidity and mortality, owing to the lack of response shown by patients to conventional therapies. Additional therapeutic targets and effective treatment options for these patients are therefore required. In the present study, a possible association of thioredoxin-interacting protein (TXNIP) with malignant glioma was evaluated. Initially, semi-quantitative and quantitative analysis of the expression levels of TXNIP in clinical specimens of primary glioma was performed via immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively, and expression levels were further correlated to the overall survival time of the patients. The proliferative, migratory and invasive properties of the glioblastoma U251 cell line, engineered to downregulate TXNIP by lentiviral transfection of a specific short hairpin RNA, were evaluated by means of in vitro assays. Consequently, IHC and RT-qPCR analysis revealed a negative association between the expression level of TXNIP and the histopathological grade of the tumor. Higher TXNIP expression level was associated with extended patient survival time. In vitro analysis revealed increased growth, migration and invasion in U251 cells with downregulated TXNIP expression compared with their non-transfected counterparts. These findings strongly indicate that TXNIP functions as a tumor suppressor in malignant glioma cells and underscore its potential as a novel therapeutic target and prognostic indicator of the condition.

MTOR controls ChREBP transcriptional activity and pancreatic β cell survival under diabetic stress.

Impaired nutrient sensing and dysregulated glucose homeostasis are common in diabetes. However, how nutrient-sensitive signaling components control glucose homeostasis and β cell survival under diabetic stress is not well understood. Here, we show that mice lacking the core nutrient-sensitive signaling component mammalian target of rapamycin (mTOR) in β cells exhibit reduced β cell mass and smaller islets. mTOR deficiency leads to a severe reduction in β cell survival and increased mitochondrial oxidative stress in chemical-induced diabetes. Mechanistically, we find that mTOR associates with the carbohydrate-response element-binding protein (ChREBP)-Max-like protein complex and inhibits its transcriptional activity, leading to decreased expression of thioredoxin-interacting protein (TXNIP), a potent inducer of β cell death and oxidative stress. Consistent with this, the levels of TXNIP and ChREBP were highly elevated in human diabetic islets and mTOR-deficient mouse islets. Thus, our results suggest that a nutrient-sensitive mTOR-regulated transcriptional network could be a novel target to improve β cell survival and glucose homeostasis in diabetes.

Hepatitis B virus X protein promotes hepatocellular carcinoma invasion and metastasis via upregulating thioredoxin interacting protein.

Hepatitis B virus X protein (HBx), a multifunctional protein encoded by the X gene of the hepatitis B virus (HBV) is involved in the metastasis of HBV-associated hepatocellular carcinoma (HCC) through various pathways, including upregulating intracellular reactive oxygen species (ROS). Thioredoxin interacting protein (TXNIP) is a key mediator of intracellular ROS, but its function in HBx-mediated metastasis of HBV-associated HCC is elusive. In the present study, HBV-associated HCC tissues with or without metastasis and HepG2 cells were used to study the function of TXNIP in HBx-mediated metastasis of HBV-associated HCC. Initially, the expression levels of TXNIP and HBx in HBV-associated HCC tissues were detected by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. The results revealed that high expression of TXNIP may be an independent risk factor for metastasis of HBV-associated HCC, and the mRNA levels of TXNIP and HBx were positively associated. Secondly, the association between HBx and TXNIP was investigated using a HBx expression stable cell line, in which HBx expression was induced and controlled by doxycycline. The results demonstrated that HBx may upregulate TXNIP expression in HepG2 cells. Thirdly, the effects of TXNIP and HBx on HepG2 cell migration and invasion were studied by scratch and Matrigel invasion assays, respectively. The results demonstrated that TXNIP overexpression enhanced HepG2 cell migration and invasion. In addition, ectopic expression of HBx promoted HepG2 cell migration and invasion, and this effect may be attenuated by knockdown of TXNIP expression, which indicated that TXNIP may be involved in the process. In summary, the present results demonstrated that TXNIP may be involved in HBx-mediated metastasis of HBV-associated HCC.

TXNIP regulates mitophagy in retinal M\xfcller cells under high-glucose conditions: implications for diabetic retinopathy

Thioredoxin-interacting protein (TXNIP) is involved in oxidative stress and apoptosis in diabetic retinopathy. However, the role of TXNIP in the removal of damaged mitochondria (MT) via mitophagy, a process of macroautophagy, remains unexplored. Here we investigate the associated cellular and molecular mechanisms underlying mitophagy in retinal cells under diabetic conditions. For this, we maintained a rat Müller cell line (rMC1) under high-glucose (25 mM, HG) or low-glucose (5.5 mM, LG) condition for 5 days. Our data reveal that HG upregulates TXNIP in the cytosol as well as in the MT. Moreover, mitochondrial oxidative stress and membrane depolarization occur under prolonged hyperglycemia leading to fragmentation. These damaged MT are targeted to lysosome for mitophagic degradation, as is evident by co-localization of mitochondrial protein COXIV, a subunit of cytochrome c oxidase, with autophagosome marker LC3BII and the lysosomal membrane protein LAMP2A. In addition, under HG conditions, there is an accumulation of dynamin-related fission protein Drp1 and E3 ubiquitin ligase Parkin in damaged MT, suggesting their roles in mitochondrial fragmentation and ubiquitination, respectively, which is absent in LG conditions. Subsequently, ubiquitin receptors, optineurin and p62/sequestrome 1, bind to the damaged MT and target them to LC3BII autophagosomes. Conversely, TXNIP knockout via CRISPR/Cas9 and TXNIP gRNA prevents the HG-induced mitochondrial damage and mitophagy in rMC1. Last, TXNIP level is also significantly upregulated in the diabetic rat retina in vivo and induces radial glial fibrillary acidic protein expression, a marker for Müller glia activation, and the formation of LC3BII puncta, which are prevented by intravitreal injection of TXNIP siRNA. Therefore, TXNIP represents a potential target for preventing ocular complications of diabetes.

Activation of NLRP3 Inflammasome by Advanced Glycation End Products Promotes Pancreatic Islet Damage.

Accumulation of advanced glycation end products (AGEs) contributes to ageing and age-related diseases, especially type 2 diabetes. The NLRP3 inflammasome, as a vital component of the innate immune system, is implicated in the pathogenesis of type 2 diabetes. However, the role of the NLRP3 inflammasome in AGE-induced pancreatic islet damage remains largely unclear. Results showed that administration of AGEs (120 mg/kg for 6 weeks) in C57BL/6J mice induced an abnormal response to glucose (as measured by glucose tolerance and insulin release), pancreatic β-cell ultrastructural lesion, and cell death. These effects were associated with an excessive superoxide anion level, significant increased protein expression levels for NADPH oxidase 2 (NOX2), thioredoxin-interacting protein (TXNIP), NLRP3, and cleaved IL-1β, enhanced caspase-1 activity, and a significant increase in the levels of TXNIP–NLRP3 protein interaction. Ablation of the NLRP3 inflammasome or treatment with antioxidant N-acetyl-cysteine (NAC) clearly ameliorated these effects. In conclusion, our results reveal a possible mechanism for AGE-induced pancreatic islet damage upon NLRP3 inflammasome activation.

Associations of polymorphisms in TXNIP and gene-environment interactions with the risk of coronary artery disease in a Chinese Han population.

Single nucleotide polymorphisms (SNPs) in thioredoxin‐interacting protein (TXNIP) gene may modulate TXNIP expression, then increase the risk of coronary artery disease (CAD). In a two‐stage case–control study with a total of 1818 CAD patients and 1963 controls, we genotyped three SNPs in TXNIP and found that the variant genotypes of SNPs rs7212 [odds ratio (OR) = 1.26, P = 0.001] and rs7211 (OR = 1.23, P = 0.005) were significantly associated with increased CAD risk under a dominant model. In haplotype analyses, compared with the reference haplotype, haplotype ‘G‐T’ had a 1.22‐fold increased risk of CAD (P = 0.003). We also observed the cumulative effects of SNPs rs7212 and rs7211 on CAD risk and the severity of coronary atherosclerosis. Moreover, the gene–environment interactions among the variant genotypes of SNP rs7212, smoking habit, alcohol drinking habit and history of type 2 diabetes were associated with a 3.70‐fold increased risk of CAD (P < 0.001). Subsequent genotype‐phenotype correlation analyses further observed the significant effects of SNP rs7212 on TXNIP mRNA expression, plasma TXNIP and malondialdehyde levels. Taken together, our data suggest that TXNIP SNPs may individually and cumulatively affect CAD risk through a possible mechanism for regulating TXNIP expression and gene–environment interactions.

Cytokines Regulate β-Cell Thioredoxin-interacting Protein (TXNIP) via Distinct Mechanisms and Pathways

Thioredoxin-interacting protein (TXNIP) is a key regulator of diabetic β-cell apoptosis and dysfunction, and TXNIP inhibition prevents diabetes in mouse models of type 1 and type 2 diabetes. Although we have previously shown that TXNIP is strongly induced by glucose, any regulation by the proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interferon γ (IFNγ) has remained largely unexplored. Moreover, even though this three-cytokine mixture is widely used to mimic type 1 diabetes in vitro, the mechanisms involved are not fully understood. Interestingly, we have now found that this cytokine mixture increases β-cell TXNIP expression; however, although TNFα had no effect, IL-1β surprisingly down-regulated TXNIP transcription, whereas IFNγ increased TXNIP levels in INS-1 β-cells and primary islets. Human TXNIP promoter analyses and chromatin immunoprecipitation studies revealed that the IL-1β effect was mediated by inhibition of carbohydrate response element binding protein activity. In contrast, IFNγ increased pro-apoptotic TXNIP post-transcriptionally via induction of endoplasmic reticulum stress, activation of inositol-requiring enzyme 1α (IRE1α), and suppression of miR-17, a microRNA that targets and down-regulates TXNIP. In fact, miR-17 knockdown was able to mimic the IFNγ effects on TXNIP, whereas miR-17 overexpression blunted the cytokine effect. Thus, our results demonstrate for the first time that the proinflammatory cytokines TNFα, IL-1β, and IFNγ each have distinct and in part opposing effects on β-cell TXNIP expression. These findings thereby provide new mechanistic insight into the regulation of TXNIP and β-cell biology and reveal novel links between proinflammatory cytokines, carbohydrate response element binding protein-mediated transcription, and microRNA signaling.

Telmisartan ameliorates oxidative stress and subarachnoid haemorrhage-induced cerebral vasospasm

Objectives: Growing evidence suggests that oxidative stress is one of the factors contributing to subarachnoid haemorrhage (SAH)-induced cerebral vasospasm. SAH-induced cerebral vasospam alters thioredoxin (Trx) cycle enzymes and thioredoxin-interacting protein (TXNIP) as an important endogenous antioxidant system. In this study, we have explored the effects of telmisartan on the vascular morphological changes, endothelial apoptosis, tissue oxidative stress status and the level of Trx cycle enzymes/ TXNIP in a rabbit SAH model.Methods: Forty male New Zealand rabbits were randomly divided into five groups of eight rabbits each: control group, sham group, SAH group, SAH + vehicle group and SAH + telmisartan group. SAH was created by a single cisterna magna blood injection. SAH + telmisartan group received telmisartan treatment (5 mg/kg intraperitoneal, once daily) for 72 h. The brainstem tissue Trx1, Trx2, Trx reductase (TrxR), TrxR1and TXNIP levels were investigated. Total oxidant status (TOS), total antioxidant status (TAS), malondialdehyde (MDA) levels and tumour necrosis factor alpha (TNF alpha) levels were investigated. Basilar artery segments were investigated for cross-sectional area, wall thickness measurements and endothelial apoptosis.Results: Telmisartan treatment restored the lowered level of Trx1, TrxR, TAS and the expression of TrxR1 seen in SAH. Telmisartan treatment also decreased TXNIP expression, TOS, MDA and TNF alpha levels. Morphological changes of cerebral vasospasm were attenuated after treatment. Endothelial apoptosis significantly reduced.Discussion: Treatment with telmisartan ameliorates oxidative stress and SAH-induced cerebral vasospasm in rabbits. These effects of telmisartan may be associated with downregulation of TXNIP and upregulation of Trx/TrxR.

High Glucose and Lipopolysaccharide Prime NLRP3 Inflammasome via ROS/TXNIP Pathway in Mesangial Cells.

While inflammation is considered a central component in the development in diabetic nephropathy, the mechanism remains unclear. The NLRP3 inflammasome acts as both a sensor and a regulator of the inflammatory response. The NLRP3 inflammasome responds to exogenous and endogenous danger signals, resulting in cleavage of procaspase-1 and activation of cytokines IL-1β, IL-18, and IL-33, ultimately triggering an inflammatory cascade reaction. This study observed the expression of NLRP3 inflammasome signaling stimulated by high glucose, lipopolysaccharide, and reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine in glomerular mesangial cells, aiming to elucidate the mechanism by which the NLRP3 inflammasome signaling pathway may contribute to diabetic nephropathy. We found that the expression of thioredoxin-interacting protein (TXNIP), NLRP3, and IL-1β was observed by immunohistochemistry in vivo. Simultaneously, the mRNA and protein levels of TXNIP, NLRP3, procaspase-1, and IL-1β were significantly induced by high glucose concentration and lipopolysaccharide in a dose-dependent and time-dependent manner in vitro. This induction by both high glucose and lipopolysaccharide was significantly inhibited by N-acetyl-L-cysteine. Our results firstly reveal that high glucose and lipopolysaccharide activate ROS/TXNIP/ NLRP3/IL-1β inflammasome signaling in glomerular mesangial cells, suggesting a mechanism by which inflammation may contribute to the development of diabetic nephropathy.

EPO Modulation of Thioredoxin Interacting Protein (TXNIP), and Key Roles for TXNIP during EPO-Dependent Human Erythroid Progenitor Cell Growth and Development

Via contemporary investigatory approaches, important new EPO action mechanisms continue to be discovered. As recent illustrating examples, lineage tracking studies have demonstrated guiding effects of EPO on the developmental fate of myeloerythroid progenitors [J Exp Med. 211:181-8]. Profiling of EPO response genes in primary erythroid progenitor cells (EPCs) has uncovered a pathway of EPO cytoprotection against leached lysosomal cathepsins in stressed erythroblasts [J Exp Med. 210:225-32]. And in a context of iron metabolism, Erythroferrone has been identified as an EPO-induced inhibitor of hepcidin [Nat Genet. 46:678-84].In a unique approach to defining EPO's cell and molecular actions, our laboratory has launched post-translational modification based LC-MS/MS proteomic analyses in human erythroid precursor cell systems. In interrogations of novel EPO/EPOR targets modified within a category of "p-TPP" motifs, TXNIP proved to be rapidly phosphorylated >10 fold due to EPO at novel C-terminal p-T349 and p-S358 sites. In parallel, multi-fold increases in TXNIP levels also were observed within 20 minutes of EPO exposure. To gain functional insight into TXNIP's roles during EPO-dependent erythropoiesis, LOF studies were performed via lentiviral shRNA mediated inhibition of TXNIP. As analyzed first in UT7epo cells, TXNIP knockdown (>90% efficiency) attenuated cell proliferation ~2.5-fold with EPO-dose dependency. This was reflected further in selective attenuation of cell cycle progression at S-phase (with only limited effects of TXNIP knockdown on cell survival observed). LOF experiments were also performed in human bone marrow derived CD34+ primary hematopoietic progenitor cells. Following initial plating, hematopoietic progenitors transduced with TXNIP shRNA exhibited a limited transient lag in growth (as compared directly with shRNA-NT transduced controls), but by day 4 of culture, and thereafter, expanded at essentially normal levels. Subsequently, erythroid progenitors with inhibited TXNIP expression prematurely committed to a program of late erythroblast differentiation. Specifically, TXNIP knockdown resulted in elevated frequencies of GPA-high erythroblasts (33.4+/-1.3% vs controls at 6.9+/-1.1%, p = 0.0001) and decreased KIT-high expression (6.4+/-3.0% vs controls at 62.5+/-7.1%, p = 0.0002). In addition, visibly obvious increases in erythroblast hemoglobinization due to TXNIP knockdown also were observed.The present investigations thus employ a unique PTM LC-MS/MS approach to identify TXNIP as a new EPO/EPOR target modified at novel C-terminal sites upon EPO expression, and in association with an indicated stabilization of TXNIP. LOF studies further reveal pro-erythropoietic roles for TXNIP as a novel mediator of EPO-dependent erythropoiesis with important effects exerted on erythroid precursor cell proliferation, together with a key requirement for TXNIP in governing a transition to a program of late erythroblast differentiation. Significance is further underlined by the nature of TXNIP as an emerging target within beta cells for small molecule inhibition in type-2 diabetics, a group highly representative among anemic chronic renal disease patients [J Diabetes Res Apr 7 2015:801348]. In particular, based on our present findings, such inhibition of TXNIP would be predicted to further compromise erythropoiesis in this CKD patient population. DisclosuresNo relevant conflicts of interest to declare.

RETRACTED: Effect of thioredoxin-interacting protein on Wnt/β-catenin signaling pathway and diabetic myocardial infarction.

To explore the regulatory role of thioredoxin-interacting protein (TXNIP) in Wnt/β-catenin signaling pathway and therefore to elucidate its function in diabetic myocardial infarction. Diabetic myocardial infarction models were generated in mice. The expression levels of TXNIP and β-catenin and level of reactive oxygen species (ROS) were determined and compared with those in control group. Human umbilical vein endothelial cells were treated with high-concentration glucose and/or silencing TXNIP and/or H2O2. After 24h, expression levels of TXNIP, β-catenin and its downstream protein Cyclin D1, and C-myc gene were determined by real-time PCR, Western blot and immunofluorescence method. The cell proliferation and ROS production capability in different groups were determined by methyl thiazolyl tetrazolium assay. Compared with control group, hyperglycemia significantly up-regulated TXNIP expression and ROS level in the myocardium and endothelial cells of myocardial infarction area, whereas the β-catenin expression was down-regulated, and the difference was statistically significant (P<0.05). In comparison with Human umbilical vein endothelial cells in the control group, high glucose level increased the levels of TXNIP expression and ROS level in cells, but reduced cell proliferation as well as migration capability and expression levels of β-catenin, Cyclin D1 and C-myc; the difference was statistically significant (P<0.05). However, this trend can be partially reversed by silencing TXNIP. Diabetic myocardial ischemia could up-regulate levels of TXNIP expression and ROS production in endothelial cells of myocardial infarction area. The regulation effect of TXNIP on β-catenin was partially achieved by changing ROS levels.

Retrospective analysis of the relationship between elevated plasma levels of TXNIP and carotid intima-media thickness in subjects with impaired glucose tolerance and early Type 2 diabetes mellitus

AimsAccelerated atherosclerosis is the major cause of mortality in diabetic patients and increased oxidative stress probably plays an important role in its development. The aim of our study was to evaluate the relationship between thioredoxin-interacting protein (TXNIP) as an oxidative stress parameter and carotid artery intima-media thickness (CIMT) as an indicator of atherosclerosis in patients with early-state diabetes and impaired glucose tolerance. MethodsThe study was a retrospective analysis of 90 patients with impaired glucose regulation (IGR), 80 patients with early Type 2 diabetes mellitus (T2DM), and 80 subjects with normal glucose tolerance (NGT) as the control group. It was conducted at the endocrine out-patient clinic and hospital department of Cangzhou Central Hospital (Cangzhou, China) from June 2012 to Oct. 2013. Plasma TXNIP was measured to evaluate the level of oxidative stress. CIMT was assessed by carotid artery ultrasonography. Soluble vascular cell adhesion molecule-1 (sVCAM-1), a risk indicator for endothelial dysfunction, was also measured. ResultsCompared to the NGT control, patients with IGR showed significantly higher plasma levels of TXNIP (P<0.05). Compared to the IGR group, patients with T2DM also had significantly higher plasma levels of TXNIP (P<0.05). CIMT was significantly higher in the subjects with abnormal glucose metabolism than in the NGT group (P<0.05). CIMT showed positive correlations with both TXNIP and sVCAM-1 levels (r=0.56 and r=0.49, respectively, both P<0.01). ConclusionsThe present study showed that plasma levels of TXNIP may be a useful predictor of subclinical atherosclerosis in Type 2 diabetic patients.

Thioredoxin-interacting protein regulates the differentiation of murine erythroid precursors

Thioredoxin-interacting protein (TXNIP) is involved in various cellular processes including redox control, metabolism, differentiation, growth, and apoptosis. With respect to hematopoiesis, TXNIP has been shown to play roles in natural killer cells, dendritic cells, and hematopoietic stem cells. Our study investigates the role of TXNIP in erythropoiesis. We observed a rapid and significant increase of TXNIP transcript and protein levels in mouse erythroleukemia cells treated with dimethyl sulfoxide or hexamethylene bisacetamide, inducers of erythroid differentiation. The upregulation of TXNIP was not abrogated by addition of the antioxidant N-acetylcysteine. The increase of TXNIP expression was confirmed in another model of erythroid differentiation, G1E-ER cells, which undergo differentiation upon activation of the GATA1 transcription factor. In addition, we showed that TXNIP levels are induced following inhibition of p38 or c-Jun N-terminal kinase (JNK)mitogen-activated protein kinases. We also observed an increase in iron uptake and a decrease in transferrin receptor protein upon TXNIP overexpression, suggesting a role in iron homeostasis. Invivo, flow cytometry analysis of cells from Txnip(-/-) mice revealed a new phenotype of impaired terminal erythropoiesis in the spleen, characterized by a partial block between basophilic and late basophilic/polychromatic erythroblasts. Based on our data, TXNIP emerges as a novel regulator of terminal erythroiddifferentiation.

The possible relationship between thioredoxin-interacting protein and the pathogenesis of type 2 diabetes mellitus

To investigate the plasma thioredoxin-interacting protein （TXNIP） levels in different glucose tolerance groups and discuss the relationship between TXNIP and insulin resistance/β-cell dysfunction in diabetes and prediabetes, and to investigate the potential relationship between TXNIP and interleukin-1β （ IL-1 β ）. According to oral glucose tolerance test, 93 participants were divided into 3 groups： diabetes mellitus group, prediabetes group, and normal glucose tolerance group. Plasma TXNIP, IL-1β, and other biochemical indices were measured. The relationship between TXNIP and glucose, IL-1 β, homeostasis model assessment for insulin resistance （HOMA-IR） , and homeostasis model assessment for β cell function （ HOMA-β ） were analyzed by using multiple linear regression techniques and Pearson＇s linear correlation analysis. Plasma TXNIP level was higher in prediabetes group compared with normal glucose tolerance group, but lower in prediabetes group compared with diabetes mellitus group [ （355.35 ±31.88 vs 274.36± 33.86,426.16 ± 63.15 ） pg/ml, P〈0.01 or P〈0.05 ]. TXNIP was positively correlated with IL-lβ and HOMA-IR, but negatively correlated with HOMA-β. Multiple linear regression analysis indicated that IL-1β exerted significant influence on TXNIP （P 〈 0. 05 ）. Plasma TXNIP level is affected by blood glucose concentration. There is a close relationship between TXNIP and IL-1 β In prediabetes patient, the TXNIP levels have already been raised. Key words: Thioredoxin-interacting protein ; Interleukin-1 β ; Diabetes mellitus, type 2 ; Insulin resistance

Minireview: Thioredoxin-interacting protein: regulation and function in the pancreatic β-cell.

Pancreatic β-cells are responsible for insulin production, and loss of functional β-cell mass is now recognized as a critical step in the pathogenesis of both type 1 and type 2 diabetes. However, the factors controlling the life and death of the pancreatic β-cell have only started to be elucidated. Discovered as the top glucose-induced gene in a human islet microarray study 12 years ago, thioredoxin-interacting protein (TXNIP) has now emerged as such a key player in pancreatic β-cell biology. Since then, β-cell expression of TXNIP has been found to be tightly regulated by multiple factors and to be dramatically increased in diabetic islets. Elevated TXNIP levels induce β-cell apoptosis, whereas TXNIP deficiency protects against type 1 and type 2 diabetes by promoting β-cell survival. TXNIP interacts with and inhibits thioredoxin and thereby controls the cellular redox state, but it also belongs to the α-arrestin family of proteins and regulates a variety of metabolic processes. Most recently, TXNIP has been discovered to control β-cell microRNA expression, β-cell function, and insulin production. In this review, the current state of knowledge regarding regulation and function of TXNIP in the pancreatic β-cell and the implications for drug development are discussed.

Hyperglycemia Regulates TXNIP/TRX/ROS Axis via p38 MAPK and ERK Pathways in Pancreatic Cancer

Approximately 85% of pancreatic cancer patients suffer from glucose intolerance or even diabetes because high glucose levels can contribute to oxidative stress which promotes tumor development. As one of the reactive oxygen species (ROS)-regulating factors, thioredoxin-interacting protein (TXNIP), is involved in the maintenance of thioredoxin (TRX)-mediated redox regulation. In this study, we demonstrated that high glucose levels increased the expression of TXNIP in time- and concentration-dependent manners and modulated the activity of TRX and ROS production in pancreatic cancer cells, BxPC-3 and Panc-1. We also found that glucose activated both p38 MAPK and ERK pathways and inhibitors of these pathways impaired the TXNIP/TRX/ROS axis. Knockdown of TXNIP restored TRX activity and decreased ROS production under high glucose conditions. Moreover, we observed that the integrated optical density (IOD) of TXNIP staining as well as the protein and mRNA expression levels of TXNIP were higher in the tumor tissues of pancreatic cancer patients with diabetes. Taken together, these results indicate that hyperglycemia-induced TXNIP expression is involved in diabetes-mediated oxidative stress in pancreatic cancer via p38 MAPK and ERK pathways.

Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

Reversing Drug Resistance and CD28 Mediated Survival In Myeloma Via The Novel Redox Regulator Thioredoxin

Despite novel chemotherapeutic regimens, myeloma patients invariably develop drug resistance and eventually die due to disease relapse, highlighting the necessity for newer therapies to extend survival. Interactions with bone marrow stroma are essential for drug resistance and survival in myeloma (MM). Not much is known about the specific cell types or molecular interactions that mediate this. Clinical studies have shown that expression of CD28 (the prototypic T cell costimulatory molecule) on MM correlates with disease progression. Our recent publication and abstracts (ASH2012 #722, ASH2011 #147, ASH2010 #132) show that interaction with dendritic cells (DC) in the bone marrow provide pro-survival signals to MM through CD28 via interactions with its ligands CD80/CD86 on DC or CD86 on other MM. Clinical studies have separately shown that CD28 or CD86 expression on MM portends poor survival in myeloma patients. In our previous ASH abstracts, we have shown that blocking MM CD28 interactions with CD80/CD86 on DC or other MM using novel reagents such as CTLA4Ig (Abatacept®, a recombinant fusion protein between CTLA4 and human IgFc) or blocking αCD28(Fab) fragments can reduce CD28 mediated survival in MM. In contrast, activating CD28 on MM with agonistic antibodies improves survival against different therapeutics or serum starvation. Another mechanism by which cancer cells survive exogenous stress is via redox regulation and our data that show that BSA (a known scavenger of reactive oxygen species (ROS)) can alleviate CTLA4Ig mediated sensitization of MM cells (Fig 1) suggest involvement of redox regulation in CD28 mediated MM survival. Intrinsic oxidative stress is a hallmark of cancer and is associated with abnormal cancer growth and progression. Others have reported that cancer cells adapt to intrinsic oxidative stress by developing enhanced anti-oxidant capacity, and are more resistant to exogenous stress. Literature also suggests that cancer cells that have higher intrinsic oxidative stress are also more likely to be sensitive to any disruption of redox regulation than normal cells. In MM, flow cytometric analysis using ROS dye CM-DCFDA show significantly higher (8-9 fold) basal ROS levels in the drug resistant U266 as opposed to the drug sensitive cell line MM.1S (Fig 2). Thioredoxin (TRX1) is a key ROS induced anti-oxidant protein essential for redox regulation and survival in many types of cancers. But not much is known about its role in myeloma survival. Interestingly, gene expression analysis of public datasets of plasma cells from normal, MGUS and myeloma patients show significant increases in the levels of TRX1 with disease progression. Further, within the myeloma patient group, TRX1 levels were significantly higher in the “relapsed” group relative to new or smoldering MM groups (Fig 3). In contrast, the levels of thioredoxin-interacting protein (TXNIP), a negative regulator of thioredoxin was significantly lower in myeloma patients and in the relapsed group, compared to normal or new diagnosed patients respectively (Fig 3). Expression trends for TRX1 and TXNIP were inversely correlated across patient groups. While CD28 activation in MM can alleviate drug induced cell death in myeloma, it could not overcome cell death induced by the specific TRX1 inhibitor PX-12 (Biomira Inc, currently under Phase 2 clinical trials for pancreatic cancer). Moreover, viability assays show that high-ROS cell line U266 was 4 fold more sensitive to PX-12 than the low-ROS MM.1S cells which is interesting since U266 is drug resistant and is thus representative of relapsed myeloma. ROS assays with PX-12 on MM show rapid dose dependent increases in ROS levels in U266 cells, while it was much lower in MM.1S suggesting a rationale for the higher sensitivity of U266. This was reversed when a ROS scavenger N-acetyl cysteine (NAC) was added. NAC also completely abrogated PX-12 mediated apoptosis in both myeloma cell lines U266 and MM.1S suggesting that PX-12's activity was solely via disruption of redox regulation. Our data reveal an important redox regulatory mechanism mediated by thioredoxin which play a supportive role for CD28 mediated survival in myeloma and disease relapse, disruption of which could selectively target relapsed drug resistant myeloma. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

FOXO1 Competes with Carbohydrate Response Element-binding Protein (ChREBP) and Inhibits Thioredoxin-interacting Protein (TXNIP) Transcription in Pancreatic Beta Cells

Thioredoxin-interacting protein (TXNIP) has emerged as an important factor in pancreatic beta cell biology, and tight regulation of TXNIP levels is necessary for beta cell survival. However, the mechanisms regulating TXNIP expression have only started to be elucidated. The forkhead boxO1 transcription factor (FOXO1) has been reported to up-regulate TXNIP expression in neurons and endothelial cells but to down-regulate TXNIP in liver, and the effects on beta cells have remained unknown. We now have found that FOXO1 binds to the TXNIP promoter in vivo in human islets and INS-1 beta cells and significantly decreases TXNIP expression. TXNIP promoter deletion analyses revealed that an E-box motif conferring carbohydrate response element-binding protein (ChREBP)-mediated, glucose-induced TXNIP expression is necessary and sufficient for this effect, and electromobility shift assays confirmed FOXO1 binding to this site. Moreover, FOXO1 blocked glucose-induced TXNIP expression and reduced glucose-induced ChREBP binding at the TXNIP promoter without affecting ChREBP expression or nuclear localization, suggesting that FOXO1 may compete with ChREBP for binding to the TXNIP promoter. In fact, a FOXO1 DNA-binding mutant (FOXO1-H215R) failed to inhibit TXNIP transcription, and the effects were not restricted to TXNIP as FOXO1 also inhibited transcription of other ChREBP target genes such as liver pyruvate kinase. Together, these results demonstrate that FOXO1 inhibits beta cell TXNIP transcription and suggest that FOXO1 confers this inhibition by interfering with ChREBP DNA binding at target gene promoters. Our findings thereby reveal a novel gene regulatory mechanism and a previously unappreciated cross-talk between FOXO1 and ChREBP, two major metabolic signaling pathways.