TXNIP is a Mediator of ER Stress-Induced β-Cell Inflammation and Apoptosis: A Dissertation

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. The pathogenesis of these diseases involves β-cell dysfunction and death. The primary function of β-cells is to tightly regulate the secretion, production, and storage of insulin in response to blood glucose levels. In order to manage insulin biosynthesis, β-cells have an elaborate endoplasmic reticulum (ER). The ER is an essential organelle for the proper processing and folding of proteins such as proinsulin. Proteins fold properly when the ER protein load balances with the ER folding capacity that handles this load. Disruption of this ER homeostasis by genetic and environmental stimuli leads to an accumulation of misfolded and unfolded proteins, a condition known as ER stress. Upon ER stress, the unfolded protein response (UPR) is activated. The UPR is a signaling network that aims to alleviate ER stress and restore ER homeostasis promoting cell survival. Hence, the UPR allows β-cells to handle the physiological fluctuations of insulin demand. However upon severe unresolvable ER stress conditions such as during diabetes progression, the UPR switches to pathological outputs leading to β-cell dysfunction and apoptosis. Severe ER stress may also trigger inflammation and accumulating evidence suggests that inflammation also contributes to β-cell failure, but the mechanisms remain elusive. In this dissertation, we demonstrate that thioredoxin interacting protein (TXNIP) mediates ER stress induced β-cell inflammation and apoptosis. During a DNA microarray analysis to identify novel survival and death components of the UPR, we identified TXNIP as an interesting proapoptotic candidate as it has been linked to glucotoxicity in β-cells. During our detailed investigation, we discovered that TXNIP is selectively expressed in β-cells of the pancreas and is strongly induced by ER stress through the IRE1α and PERK-eIF2α arms of the UPR and specifically its transcription is regulated by activating transcription factor 5 (ATF5) and carbohydrate response element binding protein (ChREBP) transcription factors. As TXNIP has been shown to activate the Nod-like receptor protein 3 (NLRP3) inflammasome leading to the production of the inflammatory cytokine interleukin-1β (IL- 1β), we hypothesized that perhaps TXNIP has a role in IL-1β production under ER stress. We show that ER stress can induce IL-1β production and that IL-1β is capable of binding to IL-1 type 1 receptor (IL-1R1) on the surface of β-cells stimulating its own expression. More importantly, we demonstrate that TXNIP does indeed play a role in ER stress mediated IL-1β production through the NLRP3 inflammasome. Furthermore, we also confirmed that TXNIP is a mediator of β-cell apoptosis under ER stress partially through IL-1β signaling. Collectively, we provide significant novel findings that TXNIP is a component of the UPR, mediates IL-1β production and autostimulation, and induces cell death under ER stress in β-cells. It is becoming clear that TXNIP has a role in the pathogenesis of diabetes and is a link between ER stress, oxidative stress and inflammation. Understanding the molecular mechanisms involved in TXNIP expression, activity, and function as we do here will shed light on potential therapeutic strategies to tackle diabetes

Valproate, a Mood Stabilizer, Induces WFS1 Expression and Modulates Its Interaction with ER Stress Protein GRP94

Valproate is a standard treatment for bipolar disorder and a first-line mood stabilizer. The molecular mechanisms underlying its actions in bipolar disorder are unclear. It has been suggested that the action of valproate is linked to changes in gene expression and induction of endoplasmic reticulum (ER) stress-response proteins.Here we show that valproate modulates the ER stress response through the regulation of WFS1, an important component for mitigating ER stress. Therapeutic concentrations of valproate induce expression of WFS1 mRNA and activate the WFS1 promoter. In addition, WFS1 forms a complex with GRP94, an ER stress-response protein, in which valproate dose-dependently enhances its dissociation from GRP94.These results suggest that the therapeutic effects of valproate in bipolar disorder may be mediated by WFS1 expression and its dissociation from GRP94

The binary switch that controls the life and death decisions of ER stressed beta cells

Diabetes mellitus is a group of common metabolic disorders defined by hyperglycemia. One of the most important factors contributing to hyperglycemia is dysfunction and death of beta cells. Increasing experimental, clinical, and genetic evidence indicates that endoplasmic reticulum (ER) stress plays an important role in beta cell dysfunction and death during the progression of type 1 and type 2 diabetes as well as genetic forms of diabetes such as Wolfram syndrome. The mechanisms of ER stress-mediated beta cell dysfunction and death are complex and not homogenous. Here we review the recent key findings on the role of ER stress and the unfolded protein response (UPR) in beta cells and the mechanisms of ER stress-mediated beta cell dysfunction and death. Complete understanding of these mechanisms will lead to novel therapeutic modalities for diabetes

A switch from life to death in endoplasmic reticulum stressed beta-cells

beta-Cell death is an important pathogenic component of both type 1 and type 2 diabetes. Recent findings indicate that cell signalling pathways emanating from the endoplasmic reticulum (ER) play an important role in the regulation of beta-cell death during the progression of diabetes. Homeostasis within the ER must be maintained to produce properly folded secretory proteins, such as insulin, in response to the body\u27s need for them. However, the sensitive protein-folding environment in the ER can be perturbed by genetic and environmental factors leading to ER stress. To counteract ER stress, beta-cells activate cell signalling pathways termed the unfolded protein response (UPR). The UPR functions as a binary switch between life and death, regulating both survival and death effectors. The outcome of this switch depends on the nature of the ER stress condition, the regulation of UPR activation and the expression and activation of survival and death components. This review discusses the mechanisms and the components in this switch and highlights the roles of this UPR\u27s balancing act between life and death in beta-cells

The binary switch between life and death of endoplasmic reticulum-stressed beta cells

PURPOSE OF REVIEW: beta-Cell death is an important pathogenic component of both type 1 and type 2 diabetes. However, the specific molecular pathways and interactions involved in this process are not completely understood. Increasing evidence indicates that a type of cell stress called endoplasmic reticulum stress (ER stress) plays an important role in beta-cell death. In the present article, we discuss a potential paradigm of ER stress-mediated beta-cell death. RECENT FINDINGS: Upon ER stress conditions, a signaling network termed the unfolded protein response (UPR) is activated. The UPR regulates adaptive effectors to attenuate ER stress and restore ER homeostasis promoting cell survival. Paradoxically the UPR also regulates apoptotic effectors. When adaptive effectors fail to attenuate ER stress, these apoptotic effectors take into effect leading to cell death. The nature of this switch between life and death is currently under study. SUMMARY: Depending on the nature of the stress condition, the UPR either protects beta cells or promotes their death. The mechanisms of this switch are not well understood but involve the balance between adaptive and apoptotic factors regulated by the UPR. In the present article, we review examples of this UPR balancing act between life and death and the potential mechanisms involved

Protein Arginine Methyltransferase 5 (Prmt5) Promotes Gene Expression of Peroxisome Proliferator-Activated Receptor gamma2 (PPARgamma2) and Its Target Genes during Adipogenesis

Regulation of adipose tissue formation by adipogenic-regulatory proteins has long been a topic of interest given the ever-increasing health concerns of obesity and type 2 diabetes in the general population. Differentiation of precursor cells into adipocytes involves a complex network of cofactors that facilitate the functions of transcriptional regulators from the CCATT/enhancer binding protein, and the peroxisome proliferator-activated receptor (PPAR) families. Many of these cofactors are enzymes that modulate the structure of chromatin by altering histone-DNA contacts in an ATP-dependent manner or by posttranslationally modifying the histone proteins. Here we report that inhibition of protein arginine methyltransferase 5 (Prmt5) expression in multiple cell culture models for adipogenesis prevented the activation of adipogenic genes. In contrast, overexpression of Prmt5 enhanced adipogenic gene expression and differentiation. Chromatin immunoprecipitation experiments indicated that Prmt5 binds to and dimethylates histones at adipogenic promoters. Furthermore, the presence of Prmt5 promoted the binding of ATP-dependent chromatin-remodeling enzymes and was required for the binding of PPARgamma2 at PPARgamma2-regulated promoters. The data indicate that Prmt5 acts as a coactivator for the activation of adipogenic gene expression and promotes adipogenic differentiation

Valproate increases the expression of WFS1 without inducing other ER stress markers.

<p>(A) Neuro-2a cells were treated with valproate (VPA, 100ug/ml) or lithium (Li, 1mM) for 4 hr, 24 hr, and 48 hr. Expression levels of Wfs1, phospho-eIF2α (P-eIF2α) and Actin were measured by immunoblot. The relative amounts of the proteins, Wfs1 and P-eIF2α, which are adjusted by the amount of actin, are shown in the right panels. (B) Expression levels of Wfs1, GRP94, GRP78, total Xbp-1, spliced Xbp-1, and Aatf were measured by quantitative real-time PCR (n = 3; values are mean±SD).</p

A speculative model of the action of valproate in the regulating of WFS1 and in the treatment of bipolar disorder.

<p>A speculative model of the action of valproate in the regulating of WFS1 and in the treatment of bipolar disorder.</p

WFS1 promoter is activated by valproate.

<p>SH-SY5Y cells were transfected with a reporter plasmid containing 500 bases of the WFS1 promoter driving the luciferase gene (pGL3-WFS1-long), a control reporter plasmid containing only 60 bases of the WFS1 promoter (pGL3-WFS1-short), or control plasmid (pGL3) plus XBP-1 expression plasmid or control plasmid. The cells were then treated with two different concentrations of valproate, 50 µg/ml and 200 µg/ml, for 6 hr.</p