Baseline Intrahepatic and Peripheral Innate Immunity are Associated with Hepatitis C Virus Clearance During Direct-Acting Antiviral Therapy.

Hepatitis C virus (HCV) infection induces interferon (IFN)-stimulated genes (ISGs) and downstream innate immune responses. This study investigated whether baseline and on-treatment differences in these responses predict response versus virological breakthrough during therapy with direct-acting antivirals (DAAs). Thirteen HCV genotype 1b-infected patients who had previously failed a course of pegylated IFN/ribavirin were retreated with asunaprevir/daclatasvir for 24 weeks. After pretreatment biopsy, patients were randomized to undergo a second biopsy at week 2 or 4 on therapy. Microarray and NanoString analyses were performed on paired liver biopsies and analyzed using linear mixed models. As biomarkers for peripheral IFN responses, peripheral blood natural killer cells were assessed for phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and degranulation. Nine of 13 (69%) patients achieved sustained virological response at 12 weeks off therapy (SVR12), and 4 experienced virological breakthroughs between weeks 4 and 12. Patients who achieved SVR12 displayed higher ISG expression levels in baseline liver biopsies and a higher frequency of pSTAT1 and TRAIL-expressing, degranulating natural killer cells in baseline blood samples than those who experienced virological breakthrough. Comparing gene expression levels from baseline and on-therapy biopsies, 408 genes (±1.2-fold, P < 0.01) were differentially expressed. Genes down-regulated on treatment were predominantly ISGs. Down-regulation of ISGs was rapid and correlated with HCV RNA suppression. Conclusion: An enhanced IFN signature is observed at baseline in liver and blood of patients who achieve SVR12 compared to those who experience a virological breakthrough; the findings suggest that innate immunity may contribute to clearance of HCV during DAA therapy by preventing the emergence of resistance-associated substitutions that lead to viral breakthrough during DAA therapy.

Polyamines Stimulate the Level of the σ38 Subunit (RpoS) of Escherichia coli RNA Polymerase, Resulting in the Induction of the Glutamate Decarboxylase-dependent Acid Response System via the gadE Regulon.

To study the physiological roles of polyamines, we carried out a global microarray analysis on the effect of adding polyamines to an Escherichia coli mutant that lacks polyamines because of deletions in the genes in the polyamine biosynthetic pathway. Previously, we have reported that the earliest response to polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR). We also presented preliminary evidence for the involvement of rpoS and gadE regulators. In the current study, further confirmation of the regulatory roles of rpoS and gadE is shown by a comparison of genome-wide expression profiling data from a series of microarrays comparing the genes induced by polyamine addition to polyamine-free rpoS(+)/gadE(+) cells with genes induced by polyamine addition to polyamine-free ΔrpoS/gadE(+) and rpoS(+)/ΔgadE cells. The results indicate that most of the genes in the E. coli GDAR system that are induced by polyamines require rpoS and gadE. Our data also show that gadE is the main regulator of GDAR and other acid fitness island genes. Both polyamines and rpoS are necessary for the expression of gadE gene from the three promoters of gadE (P1, P2, and P3). The most important effect of polyamine addition is the very rapid increase in the level of RpoS sigma factor. Our current hypothesis is that polyamines increase the level of RpoS protein and that this increased RpoS level is responsible for the stimulation of gadE expression, which in turn induces the GDAR system in E. coli.

Conditional knock-out reveals a requirement for O-linked N-Acetylglucosaminase (O-GlcNAcase) in metabolic homeostasis.

O-GlcNAc cycling is maintained by the reciprocal activities of the O-GlcNAc transferase and the O-GlcNAcase (OGA) enzymes. O-GlcNAc transferase is responsible for O-GlcNAc addition to serine and threonine (Ser/Thr) residues and OGA for its removal. Although the Oga gene (MGEA5) is a documented human diabetes susceptibility locus, its role in maintaining insulin-glucose homeostasis is unclear. Here, we report a conditional disruption of the Oga gene in the mouse. The resulting homozygous Oga null (KO) animals lack OGA enzymatic activity and exhibit elevated levels of the O-GlcNAc modification. The Oga KO animals showed nearly complete perinatal lethality associated with low circulating glucose and low liver glycogen stores. Defective insulin-responsive GSK3ß phosphorylation was observed in both heterozygous (HET) and KO Oga animals. Although Oga HET animals were viable, they exhibited alterations in both transcription and metabolism. Transcriptome analysis using mouse embryonic fibroblasts revealed deregulation in the transcripts of both HET and KO animals specifically in genes associated with metabolism and growth. Additionally, metabolic profiling showed increased fat accumulation in HET and KO animals compared with WT, which was increased by a high fat diet. Reduced insulin sensitivity, glucose tolerance, and hyperleptinemia were also observed in HET and KO female mice. Notably, the respiratory exchange ratio of the HET animals was higher than that observed in WT animals, indicating the preferential utilization of glucose as an energy source. These results suggest that the loss of mouse OGA leads to defects in metabolic homeostasis culminating in obesity and insulin resistance.

A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome.

Protein-O-linked N-Acetyl-ß-D-glucosaminidase (O-GlcNAcase, OGA; also known as hexosaminidase C) participates in a nutrient-sensing, hexosamine signaling pathway by removing O-linked N-acetylglucosamine (O-GlcNAc) from key target proteins. Perturbations in O-GlcNAc signaling have been linked to Alzheimer's disease, diabetes and cancer. Mammalian O-GlcNAcase exists as two major spliced isoforms differing only by the presence (OGA-L) or absence (OGA-S) of a histone-acetyltransferase domain. Here we demonstrate that OGA-S accumulates on the surface of nascent lipid droplets with perilipin-2; both of these proteins are stabilized by proteasome inhibition. We show that selective downregulation of OGA-S results in global proteasome inhibition and the striking accumulation of ubiquitinylated proteins. OGA-S knockdown increased levels of perilipin-2 and perilipin-3 suggesting that O-GlcNAc-dependent regulation of proteasomes might occur on the surface of lipid droplets. By locally activating proteasomes during maturation of the nascent lipid droplet, OGA-S could participate in an O-GlcNAc-dependent feedback loop regulating lipid droplet surface remodeling. Our findings therefore suggest a mechanistic link between hexosamine signaling and lipid droplet assembly and mobilization.

Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes.

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.

EZH1 and EZH2 promote skeletal growth by repressing inhibitors of chondrocyte proliferation and hypertrophy.

Histone methyltransferases EZH1 and EZH2 catalyse the trimethylation of histone H3 at lysine 27 (H3K27), which serves as an epigenetic signal for chromatin condensation and transcriptional repression. Genome-wide associated studies have implicated EZH2 in the control of height and mutations in EZH2 cause Weaver syndrome, which includes skeletal overgrowth. Here we show that the combined loss of Ezh1 and Ezh2 in chondrocytes severely impairs skeletal growth in mice. Both of the principal processes underlying growth plate chondrogenesis, chondrocyte proliferation and hypertrophy, are compromised. The decrease in chondrocyte proliferation is due in part to derepression of cyclin-dependent kinase inhibitors Ink4a/b, while ineffective chondrocyte hypertrophy is due to the suppression of IGF signalling by the increased expression of IGF-binding proteins. Collectively, our findings reveal a critical role for H3K27 methylation in the regulation of chondrocyte proliferation and hypertrophy in the growth plate, which are the central determinants of skeletal growth.

Disruption of O-GlcNAc cycling by deletion of O-GlcNAcase (Oga/Mgea5) changed gene expression pattern in mouse embryonic fibroblast (MEF) cells.

Adding a single O-GlcNAc moiety to a Ser/Thr molecule of a protein by O-GlcNAc transferase and transiently removing it by O-GlcNAcase is referred to as O-GlcNAc cycling (or O-GlcNAcylation). This O-GlcNAc modification is sensitive to nutrient availability and also shows cross talk with phosphorylation signaling, affecting downstream targets. A mouse model system was developed and evaluated to show genome wide transcriptional changes associated with disruption of O-GlcNAc cycling. Mouse embryonic fibroblast cells derived from O-GlcNAcase (Oga) knock out (KO), heterozygous (Het) and wild type (WT) embryos were used for an Affymetrix based microarray. Results are deposited in GEO dataset GSE52721. Data reveals that Oga KO MEFs had 2534 transcripts differentially expressed at 1.5 fold level while Oga heterozygous MEFs had 959 transcripts changed compared to WT MEFs. There were 1835 transcripts differentially expressed at 1.5 fold Het versus WT comparison group. Gene ontology analysis indicated differentially expressed genes enriched in metabolic, growth, and cell proliferation categories.

Paradoxical regulation of Sp1 transcription factor by glucagon.

Insulin is a potent regulator of Sp1 transcription factor. To examine if glucagon, which usually antagonizes insulin, regulates Sp1, we assessed the levels of Sp1 by Western blotting from H-411E cells exposed to glucagon with or without insulin. Glucagon alone (1.5 x 10(-9) to 1.5 x 10(-5) M) stimulated Sp1 accumulation but inhibited insulin's (10,000 microU/ml) stimulatory effect on Sp1. We also assessed the effect of TNF-alpha, wortmannin, a PI3K inhibitor, and cAMP-dependent protein kinase inhibitor on Sp1 accumulation. While TNF-alpha (5 ng/ml) blocked insulin-stimulated Sp1, it failed to block stimulation of Sp1 by glucagon (1.5 x 10(-5) M). Similarly, wortmannin inhibited insulin- but not glucagon-stimulated Sp1, whereas protein kinase inhibitor had an opposite effect. Thus, insulin acts primarily via PI3K, and glucagon apparently stimulates through a cAMP-dependent pathway. Insulin increased the staining intensity of Sp1 seen exclusively in the nuclei of H-411E cells. Sp1 was demonstrable in both nucleus and cytoplasm after glucagon treatment. Finally, as judged by immunoblotting to specific antibody, insulin but not glucagon, stimulated O-glycosylation of Sp1. Thus, unique signaling mechanisms mediate the response of Sp1 to glucagon in the presence or absence of insulin.

GLUT9 is differentially expressed and targeted in the preimplantation embryo.

During preimplantation development in the mouse, it is crucial that glucose metabolism not be compromised. Any decrease in glucose uptake at this stage in development can compromise the developing embryo. We have cloned another member of the glucose transporter family, GLUT9, which is expressed embryonically. Three different isoforms were identified. We have shown that two of the mouse GLUT9 isoforms transport glucose at a rate significantly greater than controls. Expression analysis of the preimplantation blastocyst identifies only the presence of the shorter GLUT9 isoform, RT-PCR and Western immunoblot confirmed this finding. A differential pattern of expression was seen with GLUT9 present at the plasma membrane in one- and two-cell zygotes and in an intracellular compartment in trophectoderm cells at a blastocyst stage. Although blocking GLUT9 expression during preimplantation development had no effect on glucose transport or apoptosis, transfer of these embryos into pseudopregnant mice resulted in increased pregnancy loss, suggesting that GLUT9 is critical for early preimplantation development.