Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 [Molecular Bases of Disease]

December 14th, 2015 by Chen, Q., Lu, M., Monks, B. R., Birnbaum, M. J.

Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, while the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was significantly decreased in livers with insulin signaling disrupted by ablation of insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

Low Level Pro-Inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide Mediated Protein Kinase C{delta} [Signal Transduction]

December 14th, 2015 by

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine cocktail containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of Connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.
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Inhibition of MAP Kinase-Interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing both a 5′-Terminal Cap and Hairpin [RNA]

December 14th, 2015 by Korneeva, N. L., Song, A., Gram, H., Edens, M. A., Rhoads, R. E.

The mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular-signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eIF4G and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5′-ends, we show that a MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5′-untranslated region (UTR). Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m7GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5′-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.
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Farnesoid X receptor protects against kidney injury in uninephrectomized obese mice [Metabolism]

December 11th, 2015 by Gai, Z., Gui, T., Hiller, C., Kullak-Ublick, G. A.

Activation of the farnesoid X receptor (FXR) has indicated a therapeutic potential for this nuclear bile acid receptor in the prevention of diabetic nephropathy and obesity-induced renal damage. Here, we investigated the protective role of FXR against kidney damage induced by obesity in mice that had undergone uninephrectomy, a model resembling the clinical situation of kidney donation by obese individuals. Mice fed a high-fat diet developed the core features of metabolic syndrome, with subsequent renal lipid accumulation and renal injury, including glomerulosclerosis, interstitial fibrosis, and albuminuria. The effects were accentuated by uninephrectomy. In human renal biopsies, staining of 4-hydroxynonenal (4-HNE), glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP), markers of ER stress, was more prominent in the proximal tubules of 15 obese compared with 16 non-obese patients. In mice treated with the FXR agonist obeticholic acid (OCA), renal injury, renal lipid accumulation, apoptosis and changes in lipid peroxidation were attenuated. Moreover, disturbed mitochondrial function was ameliorated and the mitochondrial respiratory chain recovered following OCA treatment. Culturing renal proximal tubular cells with free fatty acid (FFA) and FXR agonists showed that FXR activation protected cells from FFA-induced oxidative stress and ER stress, as denoted by a reduction in the level of ROS staining and Grp78 immunostaining, respectively. Several genes involved in glutathione metabolism were induced by FXR activation in the remnant kidney, which was consistent with a decreased glutathione disulfide / glutathione ratio. In summary, FXR activation maintains endogenous glutathione homeostasis and protects the kidney in uninephrectomized mice from obesity-induced injury.

SIRT1 Limits Adipocyte Hyperplasia Through c-Myc Inhibition [Metabolism]

December 11th, 2015 by

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hy-perplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD+-dependent protein deacetylase sirtuin-1 (SIRT1), a key regulator of mammalian metabo-lism, maintains proper metabolic functions in many tissues counteracting obesity. Here we re-port that differentiated adipocytes are hyperplas-tic when SIRT1 is stably knocked down in mouse 3T3-L1 preadipocytes. This phenotype is associ-ated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveals that the c-Myc pathway is altered to drive en-hanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Re-markably, differentiating SIRT1-silenced preadi-pocytes exhibit enhanced mitotic clonal expansion (MCE) accompanied by reduced levels of p27, as well as elevated levels of CCAAT/enhancer-binding protein beta (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and en-hanced proliferation phenotype are also found to be SIRT1-dependent in proliferating MEFs and differentiating human SW872 preadipocytes. Re-ducing both SIRT1 and c-Myc expression in 3T3-L1 simultaneously do not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. Better understanding of the molecu-lar mechanisms of adipocyte hyperplasia will open new venues towards understanding obesity.

Selective Recognition of H3.1K36 dimethylation / H4K16 acetylation facilitates the regulation of ATRA-responsive genes by putative chromatin reader ZMYND8 [Cell Biology]

December 11th, 2015 by

ZMYND8, a newly identified component of transcriptional coregulator network, was found to interact with Nucleosome Remodelling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting NuRD complex to damaged chromatin for repressing transcription and promoting double-strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with selective epigenetic marks H3.1K36Me2/H4K16Ac. Further, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the All Trans Retinoic Acid (ATRA)-responsive ones, through its modified histone binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation competent RNA Polymerase II phosphorylated at Ser5 in a DNA template dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone binding ability.
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Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis are Correlated with Pathogenicity and Carriage [Lipids]

December 11th, 2015 by

The degree of phosphorylation and phosphoethanolaminylation of lipid A on Neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of N. meningitidis without disease. Invasive strains were classified as Groups 1, 2 and 3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with 3 phosphates and 1 phosphoethanolamine were detected in all LOS analyzed. LOS from Groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared to LOS from Group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared to carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multi-faceted effects on homeostatic innate immune responses which have critical impact on the pathophysiology of meningococcal infections.
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Progesterone-induced acrosome exocytosis requires sequential involvement of calcium-independent iPLA2[beta] and group X sPLA2 [Developmental Biology]

December 11th, 2015 by

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR) but the molecular identity of PLA2 has remained elusive. Here we have tested the role of two intracellular (iPLA2β and cPLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2β is critical for spontaneous AR while both iPLA2β and group X sPLA2 are involved in P4-induced AR. cPLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 and Ca2+ pathways to achieve AR. At low P4 concentration, sperm achieving early AR (0-5 min post-P4) rely on iPLA2β, whereas sperm achieving late AR (20-30 min post-P4) rely on both iPLA2β and group X sPLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, PLA2s being key actors at low physiological P4 concentrations (≤ 2 μM) but not at higher P4 concentrations (>10 μM).
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The insect peptide CopA3 increases colonic epithelial cell proliferation and mucosal barrier function to prevent inflammatory responses in the gut [Cell Biology]

December 11th, 2015 by

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. Epidermal growth factor (EGF) enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide, CopA3, promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin A-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the DSS-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21Cip1/Waf1, and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21Cip1/Waf1. Taken together, our findings indicate that the insect peptide, CopA3, prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.
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Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacteylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B Cell Acute Lymphoblastic Leukemia [Signal Transduction]

December 11th, 2015 by

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements (UREs) of its target genes and regulates their transcription via chromatin remodeling, Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 tri-methylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the URE of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic Casein Kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of H3K27me3 and decreased H3K9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. Presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.
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