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Background: Dietary fructose can rapidly cause fatty liver in animals through de novo lipogenesis (DNL) and contribute to the development and severity of nonalcoholic fatty liver disease (NAFLD). In response to diverse cellular insults including endoplasmic reticulum (ER) and oxidative stress, phosphorylation of the eukaryotic translation initiation factor 2 alpha subunit (eIF2α) attenuates general translation initiation, allowing cells to conserve resources and initiate adaptive gene expression to restore homeostasis. The present study aimed to investigate the role of eIF2α phosphorylation in protecting against NAFLD induced by high fructose ingestion in a hepatocyte-specific eIF2α-phosphorylation-deficient mouse model. Methods: Hepatocyte-specific non-phosphorylatable (S51A) eIF2α knock-in (A/A;fTg/0;CreHep/0, A/AHep) mice were generated by crossing A/A;fTg/fTg mice with the floxed WT eIF2α transgene (fTg) with Alfp-Cre recombinase transgenic S/A; CreHep/0 (S/A-CreHep) mice. Hepatocyte-specific eIF2α-phosphorylation-deficient 3-month-old mice or 12-month-old mice were fed a 60% high fructose diet (HFrD) for 16 or 5 wks, and the effects of eIF2α-phosphorylation deficiency on NADP/ NADPH and GSSG/GSH levels, ROS-defense gene expression, oxidative damage, cell death, and fibrosis were observed. Results: Prolonged fructose feeding to mice caused dysregulation of the unfolded protein response (UPR) sensor activation and UPR gene expression, and then led to decreased expression of several ROS defense genes including glutathione biogenesis genes. Nonetheless, these changes were not sufficient to induce the death of eIF2α phosphorylation-sufficient hepatocytes. However, there was a substantial increase in hepatocyte death and liver fibrosis in fructose-fed middle-aged mice deficient in hepatocyte-specific eIF2α phosphorylation because of diminished antioxidant capacity due to reduced expression of antioxidant enzymes (GPX1 and HO-1) and lower NADPH and glutathione levels, as well as a possible increase in ROS-induced damage from infiltrating NOX2-expressing leukocytes; all this led to a vicious cycle of hepatocyte death and leukocyte infiltration. Conclusion: Our findings suggest that eIF2α phosphorylation maintains NADPH and GSH levels and controls the expression of ROS-defense genes, thereby protecting hepatocytes from oxidative stresses induced by fructose metabolism. [ABSTRACT FROM AUTHOR]

Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58IPK/DnajC3, an ER luminal co-chaperone. P58IPK-/- mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58IPK-/- mice, suggesting P58IPK deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58IPK-/- mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes. [ABSTRACT FROM AUTHOR]

Efficient production of insulin in response to changes in glucose levels has been a major issue for insulin gene therapy to treat diabetes. To express target genes in response to glucose specifically in hepatocytes, we generated a synthetic promoter library containing hepatocyte nuclear factor-1, CAAT/enhancer-binding protein (C/EBP) response element, and glucose-response element. Combinations of these three cis-elements in 3-, 6-, or 9-element configurations were screened for transcriptional activity and then glucose responsiveness in vitro. The most effective promoter (SP23137) was selected for further study. Intravenous administration of a recombinant adenovirus expressing furin-cleavable rat insulin under control of the SP23137 promoter into streptozotocin (STZ)-induced diabetic mice resulted in normoglycemia, which was maintained for >30 days. Glucose tolerance tests showed that treated mice produced insulin in response to glucose and cleared exogenous glucose from the blood in a manner similar to nondiabetic control mice, although the clearance was somewhat delayed. Insulin expression was seen specifically in the liver and not in other organs. These observations indicate the potential of this synthetic, artificial promoter to regulate glucose-responsive insulin production and remit hyperglycemia, thus providing a new method of liver-directed insulin gene therapy for type 1 diabetes. [ABSTRACT FROM AUTHOR]

To investigate whether the local production of glucagon-like peptide-1 (GLP-1) in the intestine can differentiate intestinal stem/progenitor cells into insulin-producing cells, we intra-intestinally injected a recombinant adenovirus expressing GLP-1 (rAd-GLP-1) into diabetic mice. There were no significant differences in body weight or food intake between rAd-GLP-1- and rAd-βGAL-treated control mice. rAd-GLP-1-treated mice showed intestinal insulin mRNA expression, insulin- and glucagon-positive cells in the intestine, and significantly increased serum insulin, but not glucagon. rAd-GLP-1 injection significantly reduced blood glucose levels and improved glucose tolerance compared with controls. Expression of transcription factors related to β cell differentiation, neurogenin3 (ngn3) and neurogenin differentiation factor (NeuroD), was detected in the intestine at 2 weeks after rAd-GLP-1 injection. We suggest that expression of GLP-1 in the intestine by intra-intestinal delivery of rAd-GLP-1 may induce differentiation of intestinal stem/progenitor cells into insulin-producing cells, mediated by ngn3 and NeuroD expression, contributing to lowered blood glucose levels in diabetic mice. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic β-cells. Expression of insulin in non-β-cells to create β-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic β-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate β-cells for possible therapeutic use for the treatment of type 1 diabetes.Molecular Therapy (2007) 15 6, 1195–1202. doi:10.1038/sj.mt.6300117 [ABSTRACT FROM AUTHOR]

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms--unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is a complex metabolic disorder characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency by β-cell failure. Even if the mechanisms underlying the pathogenesis of β-cell failure are still under investigation, recent increasing genetic, experimental, and clinical evidence indicate that hyperactivation of the unfolded protein response (UPR) to counteract metabolic stresses is closely related to β-cell dysfunction and apoptosis. Signaling pathways of the UPR are "a double-edged sword" that can promote adaptation or apoptosis depending on the nature of the ER stress condition. In this paper, we summarized our current understanding of the mechanisms and components related to ER stress in the β-cell pathogenesis of type 2 diabetes. [ABSTRACT FROM AUTHOR]

This work reports on a mechanism for irreversible resistive switching (RS) transformation from bipolar to unipolar RS behavior in SrRuO3(SRO)/Cr-doped SrZrO3 (SZO:Cr)/Pt capacitor structures prepared on a Ti/SiO2/Si substrate. Counter-clockwise bipolar RS memory current-voltage (I-V) characteristics are observed within the RS voltage window of −2.5 to +1.9 V, with good endurance and retention properties. As the bias voltage increases further beyond 4 V under a forward bias, a forming process occurs resulting in irreversible RS mode transformation from bipolar to unipolar mode. This switching mode transformation is a direct consequence of thermally activated Ti out-diffusion from a Ti adhesion layer. Transition metal Ti effectively out-diffuses through the loose Pt electrode layer at high substrate temperatures, leading to the unintended formation of a thin titanium oxide (TiOx where x < 2) layer between the Pt electrode and the SZO:Cr layer as well as additional Ti atoms in the SZO:Cr layer. Cross-sectional scanning electron microscopy, transmission electron microscopy and Auger electron spectroscopy depth-profile measurements provided apparent evidence of the Ti out-diffusion phenomenon. We propose that the out-diffusion-induced additional Ti atoms in the SZO:Cr layer contributes to the creation of the metallic filamentary channels. [ABSTRACT FROM AUTHOR]

Cellular phosphorylation events during viral infection are necessary for effective viral replication. Encephalomyocarditis (EMC) virus has been used for studies on the molecular mechanisms of viral replication, but little is known about the cellular signaling pathways involved. This investigation was initiated to determine whether mitogen-activated protein kinases (MAPKs), which are central components of signal transduction pathways in the regulation of cell proliferation, play a role in the replication of EMC virus. We examined the phosphorylation of MAPKs, including extracellular signal-regulated kinase (ERK½), p35 MAPK, and stress activated protein kinase 1/c-Jun NH[sub 2]-terminal kinase (SAPK/JNK) in EMC virus-infected L929 cells and found that p38 MAPK and SAPK-JNK, but not ERK½, were activated during viral infection. We then examined the effect of these kinases on the replication of EMC virus in L929 cells by using specific inhibitors, including genistein or herbimycin A for tyrosine kinase, SB203580 or SB202190 for p38 MAPK, and PD98059 for ERK½. We found that the tyrosine kinase and p38 MAPK inhibitors, but not the ERK½ inhibitor, suppressed viral replication and that the inhibitory effect was primarily on viral protein synthesis. Finally, we examined whether p38 MAPK is involved in the translation of EMC viral transcripts by using L929 cells transfected with a gene construct containing the internal ribosomal entry site (IRES) of EMC virus and a luciferase reporter gene. We found that the p38 MAPK inhibitor suppressed the translation of EMC viral RNA. On the basis of these observations, we conclude that p38 MAPK plays a critical role in the replication of EMC virus, probably in the translation of viral RNA. [ABSTRACT FROM AUTHOR]