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beta Actin antibody - Loading Control

RRID:AB_2305186

Antibody ID

AB_2305186

Target Antigen

beta Actin antibody - Loading Control hamster, mouse, xenopus/amphibian, rabbit, guinea pig, zebrafish/fish, porcine, bovine, sheep, canine, human, non-human primate, rat, chicken/bird, human, mouse, rat, chicken, chinese hamster, cow, dog, fish, guinea pig, monkey, pig, rabbit, rhesus monkey, sheep, xenopus laevis

Proper Citation

(Abcam Cat# ab8227, RRID:AB_2305186)

Clonality

polyclonal antibody

Comments

validation status unknown, seller recommendations provided in 2012: ELISA, ICC, ICC/IF, IHC-Fr, IHC-FrFl, IHC-P, IP, WB; Immunofluorescence; Immunohistochemistry; ELISA; Immunohistochemistry - frozen; Immunoprecipitation; Immunocytochemistry; Western Blot; Immunohistochemistry - fixed

Host Organism

rabbit

Vendor

Abcam

Hippocampal NF-κB accounts for stress-induced anxiety behaviors via enhancing nNOS-CAPON-Dexras1 coupling.

  • Zhu LJ
  • J. Neurochem.
  • 2018 Jun 2

Literature context:


Abstract:

Anxiety disorders are associated with a high social burden worldwide. Recently, increasing evidence suggests that nuclear factor kappa B (NF-κB) has significant implications for psychiatric diseases, including anxiety and depressive disorders. However, the molecular mechanisms underlying the role of NF-κB in stress-induced anxiety behaviors are poorly understood. In this study, we show that chronic mild stress (CMS) and glucocorticoids dramatically increased the expression of NF-κB subunits p50 and p65, phosphorylation and acetylation of p65, and the level of nuclear p65 in vivo and in vitro, implicating activation of NF-κB signaling in chronic stress-induced pathological processes. Using the novelty-suppressed feeding (NSF) and elevated-plus maze (EPM) tests, we found that treatment with pyrrolidine dithiocarbamate (PDTC; intrahippocampal infusion), an inhibitor of NF-κB, rescued the CMS- or glucocorticoid-induced anxiogenic behaviors in mice. Microinjection of PDTC into the hippocampus reversed CMS-induced upregulation of neuronal nitric oxide synthase (nNOS), carboxy-terminal PDZ ligand of nNOS (CAPON) and dexamethasone-induced ras protein 1 (Dexras1) and dendritic spine loss of DG granule cells. Moreover, overexpression of CAPON by infusing LV-CAPON-L-GFP into the hippocampus induced nNOS-Dexras1 interaction and anxiety-like behaviors, and inhibition of NF-κB by PDTC reduced the LV-CAPON-L-GFP-induced increases in nNOS-Dexras1 complex and anxiogenic-like effects in mice. These findings indicate that hippocampal NF-κB mediates anxiogenic behaviors, probably via regulating the association of nNOS-CAPON-Dexras1, and uncover a novel approach to the treatment of anxiety disorders. This article is protected by copyright. All rights reserved.

Funding information:
  • NIGMS NIH HHS - GM GM69373(United States)

An Intermediate Pluripotent State Controlled by MicroRNAs Is Required for the Naive-to-Primed Stem Cell Transition.

  • Du P
  • Cell Stem Cell
  • 2018 Jun 1

Literature context:


Abstract:

The embryonic stem cell (ESC) transition from naive to primed pluripotency is marked by major changes in cellular properties and developmental potential. ISY1 regulates microRNA (miRNA) biogenesis, yet its role and relevance to ESC biology remain unknown. Here, we find that highly dynamic ISY1 expression during the naive-to-primed ESC transition defines a specific phase of "poised" pluripotency characterized by distinct miRNA and mRNA transcriptomes and widespread poised cell contribution to mouse chimeras. Loss- and gain-of-function experiments reveal that ISY1 promotes exit from the naive state and is necessary and sufficient to induce and maintain poised pluripotency, and that persistent ISY1 overexpression inhibits the transition from the naive to the primed state. We identify a large subset of ISY1-dependent miRNAs that can rescue the inability of miRNA-deficient ESCs to establish the poised state and transition to the primed state. Thus, dynamic ISY1 regulates poised pluripotency through miRNAs to control ESC fate.

Funding information:
  • NCI NIH HHS - CA78412(United States)
  • NIGMS NIH HHS - R01 GM086386()

A LINE1-Nucleolin Partnership Regulates Early Development and ESC Identity.

  • Percharde M
  • Cell
  • 2018 Jun 19

Literature context:


Abstract:

Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.

Funding information:
  • NCI NIH HHS - P30 CA54174(United States)
  • NIGMS NIH HHS - R01 GM113014()
  • NIGMS NIH HHS - R01 GM123556()

iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE.

  • Oikonomidi I
  • Elife
  • 2018 Jun 13

Literature context:


Abstract:

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.

Funding information:
  • Deutsche Forschungsgemeinschaft - Emmy Noether scholarship DFG STE2310/1-1()
  • European Cooperation in Science and Technology - BM1406()
  • European Molecular Biology Organization - Installation Grant no. 2329()
  • European Regional Development Fund - CZ.2.16/3.1.00/24016()
  • Fundação Calouste Gulbenkian - 91/BD/14()
  • Fundação para a Ciência e a Tecnologia - 91/BD/14()
  • Fundação para a Ciência e a Tecnologia - BEX-BCM/3015/2014()
  • Fundação para a Ciência e a Tecnologia - LISBOA-01-0145-FEDER-031330()
  • Fundação para a Ciência e a Tecnologia - PTDC/BEX-BCM/3015/2014()
  • Fundação para a Ciência e a Tecnologia - SFRH/ BPD/117216/2016()
  • Fundação para a Ciência e a Tecnologia - SFRH/BCC/52507/2014()
  • Ministerstvo Školství, Mládeže a T?lovýchovy - LO1302()
  • Ministry of Education, Youth and Sports of the Czech Republic - LO1302()
  • NIGMS NIH HHS - R01 GM070743(United States)
  • Science Foundation Ireland - 14/IA/2622()
  • Seventh Framework Programme - Marie Curie Career Integration Grant (project no. 618769()
  • Worldwide Cancer Research - 14-1289()

[18F]FMPEP-d2 PET imaging shows age- and genotype-dependent impairments in the availability of cannabinoid receptor 1 in a mouse model of Alzheimer's disease.

  • Takkinen JS
  • Neurobiol. Aging
  • 2018 May 18

Literature context:


Abstract:

Contradictory findings on the role of the type 1 cannabinoid receptor (CB1R) during the pathogenesis of Alzheimer's disease (AD) have been reported. Here, we evaluated the CB1R brain profile in an AD mouse model using longitudinal positron emission tomography with an inverse agonist for CB1R, [18F]FMPEP-d2. APP/PS1-21 and wild-type (n = 8 in each group) mice were repeatedly imaged between 6 to 15 months of age, accompanied by brain autoradiography, western blot, and CB1R immunohistochemistry with additional mice. [18F]FMPEP-d2 positron emission tomography demonstrated lower (p < 0.05) binding ratios in the parietotemporal cortex and hippocampus of APP/PS1-21 mice compared with age-matched wild-type mice. Western blot demonstrated no differences between APP/PS1-21 and wild-type mice in the CB1R abundance, whereas significantly lower (p < 0.05) receptor expression was observed in male than female mice. The results provide the first demonstration that [18F]FMPEP-d2 is a promising imaging tool for AD research in terms of CB1R availability, but not expression. This finding may further facilitate the development of novel therapeutic approaches based on endocannabinoid regulation.

Funding information:
  • NIGMS NIH HHS - R01 GM077668-03S1(United States)

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.

  • Haider A
  • Dev. Cell
  • 2018 May 21

Literature context:


Abstract:

Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.

Funding information:
  • Canadian Institutes of Health Research - MOP-312268(Canada)

Complement C3 Drives Autophagy-Dependent Restriction of Cyto-invasive Bacteria.

  • Sorbara MT
  • Cell Host Microbe
  • 2018 May 9

Literature context:


Abstract:

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.

Funding information:
  • NIDA NIH HHS - R03 DA029480(United States)

Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

  • Mendez-Bermudez A
  • Mol. Cell
  • 2018 May 3

Literature context:


Abstract:

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer.

Funding information:
  • Medical Research Council - (United Kingdom)

Intracellular Proteolysis of Progranulin Generates Stable, Lysosomal Granulins that Are Haploinsufficient in Patients with Frontotemporal Dementia Caused by GRN Mutations.

  • Holler CJ
  • eNeuro
  • 2018 May 3

Literature context:


Abstract:

Homozygous or heterozygous mutations in the GRN gene, encoding progranulin (PGRN), cause neuronal ceroid lipofuscinosis (NCL) or frontotemporal dementia (FTD), respectively. NCL and FTD are characterized by lysosome dysfunction and neurodegeneration, indicating PGRN is important for lysosome homeostasis in the brain. PGRN is trafficked to the lysosome where its functional role is unknown. PGRN can be cleaved into seven 6-kDa proteins called granulins (GRNs); however, little is known about how GRNs are produced or if levels of GRNs are altered in FTD-GRN mutation carriers. Here, we report the identification and characterization of antibodies that reliably detect several human GRNs by immunoblot and immunocytochemistry. Using these tools, we find that endogenous GRNs are present within multiple cell lines and are constitutively produced. Further, extracellular PGRN is endocytosed and rapidly processed into stable GRNs within lysosomes. Processing of PGRN into GRNs is conserved between humans and mice and is modulated by sortilin expression and mediated by cysteine proteases (i.e. cathpesin L). Induced lysosome dysfunction caused by alkalizing agents or increased expression of transmembrane protein 106B (TMEM106B) inhibit processing of PGRN into GRNs. Finally, we find that multiple GRNs are haploinsufficient in primary fibroblasts and cortical brain tissue from FTD-GRN patients. Taken together, our findings raise the interesting possibility that GRNs carry out critical lysosomal functions and that loss of GRNs should be explored as an initiating factor in lysosomal dysfunction and neurodegeneration caused by GRN mutations.

High-intensity treadmill running impairs cognitive behavior and hippocampal synaptic plasticity of rats via activation of inflammatory response.

  • Sun LN
  • J. Neurosci. Res.
  • 2018 Apr 17

Literature context:


Abstract:

Although appropriate exercise is beneficial for enhancing brain functions, high-intensity exercise (HIE)-induced cognitive dysfunction is causing more and more concerns nowadays. In the present study, we observed the effects of high-intensity treadmill running on the spatial learning of the adult Sprague Dawley male rats in Y-maze (n = 16 per group), and investigated its possible electrophysiological and molecular mechanisms by examining in vivo hippocampal long-term potentiation (LTP), central inflammatory responses, and JNK/p38/ERK signal pathway. The Y-maze active avoidance test showed that high-intensity treadmill running impaired spatial learning ability of rats, with increased error times and prolonged training time in recognizing safety condition. Associated with the cognitive dysfunction, the induction and maintenance of hippocampal LTP were also impaired by the HIE. Furthermore, accompanied by elevated levels of inflammatory factors IL-1β, TNF-α, and iNOS, overactivation of microglia and astrocytes was also found in the CA1 region of hippocampus in the excessive exercise group, indicating an inflammatory response induced by HIE. In addition, Western blot assay showed that the phosphorylation of JNK/p38/ERK proteins was enhanced in the exercise group. These results suggest that exercise stress-induced neuronal inflammatory responses in the hippocampus are associated with HIE-induced cognitive deficits, which may be involved in the upregulation of the JNK/p38/ERK pathway. © 2016 Wiley Periodicals, Inc.

The Transcriptionally Permissive Chromatin State of Embryonic Stem Cells Is Acutely Tuned to Translational Output.

  • Bulut-Karslioglu A
  • Cell Stem Cell
  • 2018 Mar 1

Literature context:


Abstract:

A permissive chromatin environment coupled to hypertranscription drives the rapid proliferation of embryonic stem cells (ESCs) and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ESCs. The results revealed that cellular growth pathways, most prominently translation, perpetuate the euchromatic state and hypertranscription of ESCs. Acute inhibition of translation rapidly depletes euchromatic marks in mouse ESCs and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Translation inhibition promotes rewiring of chromatin accessibility, which decreases at a subset of active developmental enhancers and increases at histone genes and transposable elements. Proteome-scale analyses revealed that several euchromatin regulators are unstable proteins and continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription, and translation sets the pace of proliferation at peri-implantation and may be employed by other stem/progenitor cells.

Funding information:
  • NICHD NIH HHS - F30 HD093116()
  • NIGMS NIH HHS - R01 GM113014()
  • NIGMS NIH HHS - R01 GM123556()
  • NIGMS NIH HHS - R01 GM55040(United States)

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential.

  • Judson RN
  • Cell Stem Cell
  • 2018 Feb 1

Literature context:


Abstract:

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of β-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease.

Funding information:
  • BLRD VA - I01 BX002324()
  • NCI NIH HHS - R01 CA073808(United States)
  • NIA NIH HHS - P01 AG036695()
  • NIAMS NIH HHS - R21 AR071039()
  • RRD VA - I01 RX001222()

The Glycosyltransferase EOGT Regulates Adropin Expression in Decidualizing Human Endometrium.

  • Muter J
  • Endocrinology
  • 2018 Feb 1

Literature context:


Abstract:

In pregnancy, resistance of endometrial decidual cells to stress signals is critical for the integrity of the fetomaternal interface and, by extension, survival of the conceptus. O-GlcNAcylation is an essential posttranslational modification that links glucose sensing to cellular stress resistance. Unexpectedly, decidualization of primary endometrial stromal cells (EnSCs) was associated with a 60% reduction in O-linked β-N-acetylglucosamine (O-GlcNAc)‒modified proteins, reflecting downregulation of the enzyme that adds O-GlcNAc to substrates (O-GlcNAc transferase; OGT) but not the enzyme that removes the modification (O-GlcNAcase). Notably, epidermal growth factor domain-specific O-linked GlcNAc transferase (EOGT), an endoplasmic reticulum-specific OGT that modifies a limited number of secreted and membrane proteins, was markedly induced in differentiating EnSCs. Knockdown of EOGT perturbed a network of decidual genes involved in multiple cellular functions. The most downregulated gene upon EOGT knockdown in decidualizing cells was the energy homeostasis-associated gene (ENHO), which encodes adropin, a metabolic hormone involved in energy homeostasis and glucose and fatty acid metabolism. Analysis of midluteal endometrial biopsies revealed an inverse correlation between endometrial EOGT and ENHO expression and body mass index. Taken together, our findings revealed that obesity impairs the EOGT-adropin axis in decidual cells, which in turn points toward a mechanistic link between metabolic disorders and adverse pregnancy outcome.

Funding information:
  • NIDDK NIH HHS - R01DK-62388(United States)

Removal of prolyl oligopeptidase reduces alpha-synuclein toxicity in cells and in vivo.

  • Svarcbahs R
  • Sci Rep
  • 2018 Jan 24

Literature context:


Abstract:

Prolyl oligopeptidase (PREP) inhibition by small-molecule inhibitors can reduce alpha-synuclein (aSyn) aggregation, a key player in Parkinson's disease pathology. However, the significance of PREP protein for aSyn aggregation and toxicity is not known. We studied this in vivo by using PREP knock-out mice with viral vector injections of aSyn and PREP. Animal behavior was studied by locomotor activity and cylinder tests, microdialysis and HPLC were used to analyze dopamine levels, and different aSyn forms and loss of dopaminergic neurons were studied by immunostainings. Additionally, PREP knock-out cells were used to characterize the impact of PREP and aSyn on autophagy, proteasomal system and aSyn secretion. PREP knock-out animals were nonresponsive to aSyn-induced unilateral toxicity but combination of PREP and aSyn injections increased aSyn toxicity. Phosphorylated p129, proteinase K resistant aSyn levels and tyrosine hydroxylase positive cells were decreased in aSyn and PREP injected knock-out animals. These changes were accompanied by altered dopamine metabolite levels. PREP knock-out cells showed reduced response to aSyn, while cells were restored to wild-type cell levels after PREP overexpression. Taken together, our data suggests that PREP can enhance aSyn toxicity in vivo.

Funding information:
  • National Cancer Institute - CA163820A1(United States)

NECAPs are negative regulators of the AP2 clathrin adaptor complex.

  • Beacham GM
  • Elife
  • 2018 Jan 18

Literature context:


Abstract:

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2.

Funding information:
  • National Science Foundation - Graduate Research Fellowship DGE-1650441()
  • NIAID NIH HHS - AI082673(United States)
  • NIGMS NIH HHS - T32 GM007273()
  • NIH HHS - S10 OD018516()
  • NIH HHS - Training Grant GM007273-43()

System-wide Benefits of Intermeal Fasting by Autophagy.

  • Martinez-Lopez N
  • Cell Metab.
  • 2017 Dec 5

Literature context:


Abstract:

Autophagy failure is associated with metabolic insufficiency. Although caloric restriction (CR) extends healthspan, its adherence in humans is poor. We established an isocaloric twice-a-day (ITAD) feeding model wherein ITAD-fed mice consume the same food amount as ad libitum controls but at two short windows early and late in the diurnal cycle. We hypothesized that ITAD feeding will provide two intervals of intermeal fasting per circadian period and induce autophagy. We show that ITAD feeding modifies circadian autophagy and glucose/lipid metabolism that correlate with feeding-driven changes in circulating insulin. ITAD feeding decreases adiposity and, unlike CR, enhances muscle mass. ITAD feeding drives energy expenditure, lowers lipid levels, suppresses gluconeogenesis, and prevents age/obesity-associated metabolic defects. Using liver-, adipose-, myogenic-, and proopiomelanocortin neuron-specific autophagy-null mice, we mapped the contribution of tissue-specific autophagy to system-wide benefits of ITAD feeding. Our studies suggest that consuming two meals a day without CR could prevent the metabolic syndrome.

Funding information:
  • NCI NIH HHS - P30 CA013330()
  • NIA NIH HHS - P01 AG031782()
  • NIA NIH HHS - P30 AG038072()
  • NIA NIH HHS - R01 AG043517()
  • NIA NIH HHS - R37 AG018381()
  • NIA NIH HHS - T32 AG023475()
  • NIDDK NIH HHS - P30 DK020541()
  • NIDDK NIH HHS - P30 DK026687()
  • NIDDK NIH HHS - P30 DK041296()
  • NIDDK NIH HHS - R01 DK033823()
  • NIDDK NIH HHS - R01 DK105441()
  • NIMH NIH HHS - P50 MH096890(United States)

Organelle Specific O-Glycosylation Drives MMP14 Activation, Tumor Growth, and Metastasis.

  • Nguyen AT
  • Cancer Cell
  • 2017 Nov 13

Literature context:


Abstract:

Cancers grow within tissues through molecular mechanisms still unclear. Invasiveness correlates with perturbed O-glycosylation, a covalent modification of cell-surface proteins. Here, we show that, in human and mouse liver cancers, initiation of O-glycosylation by the GALNT glycosyl-transferases increases and shifts from the Golgi to the endoplasmic reticulum (ER). In a mouse liver cancer model, expressing an ER-targeted GALNT1 (ER-G1) massively increased tumor expansion, with median survival reduced from 23 to 10 weeks. In vitro cell growth was unaffected, but ER-G1 strongly enabled matrix degradation and tissue invasion. Unlike its Golgi-localized counterpart, ER-G1 glycosylates the matrix metalloproteinase MMP14, a process required for tumor expansion. Together, our results indicate that GALNTs strongly promote liver tumor growth after relocating to the ER.

Selective Inhibition of FOXO1 Activator/Repressor Balance Modulates Hepatic Glucose Handling.

  • Langlet F
  • Cell
  • 2017 Nov 2

Literature context:


Abstract:

Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and inhibition of glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of glucokinase is unknown, and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here, we report that SIN3A is the insulin-sensitive FOXO1 corepressor of glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of glucokinase without affecting other FOXO1 target genes and lowers glycemia without concurrent steatosis. To extend this work, we executed a small-molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.

Funding information:
  • NHLBI NIH HHS - R01 HL125649()
  • NIAAA NIH HHS - R01 AA023416()
  • NICHD NIH HHS - 5T32-HD007520(United States)
  • NIDDK NIH HHS - P30 DK026687()
  • NIDDK NIH HHS - P30 DK063608()
  • NIDDK NIH HHS - R01 DK057539()
  • NIDDK NIH HHS - R01 DK058282()
  • NIDDK NIH HHS - R01 DK064819()
  • NIDDK NIH HHS - R37 DK058282()
  • NIDDK NIH HHS - R56 DK083658()
  • NIDDK NIH HHS - T32 DK007328()

Late rDNA Condensation Ensures Timely Cdc14 Release and Coordination of Mitotic Exit Signaling with Nucleolar Segregation.

  • de Los Santos-Velázquez AI
  • Curr. Biol.
  • 2017 Nov 6

Literature context:


Abstract:

The nucleolus plays a pivotal role in multiple key cellular processes. An illustrative example is the regulation of mitotic exit in Saccharomyces cerevisiae through the nucleolar sequestration of the Cdc14 phosphatase. The peculiar structure of the nucleolus, however, has also its drawbacks. The repetitive nature of the rDNA gives rise to cohesion-independent linkages whose resolution in budding yeast requires the Cdc14-dependent inhibition of rRNA transcription, which facilitates condensin accessibility to this locus. Thus, the rDNA condenses and segregates later than most other yeast genomic regions. Here, we show that defective function of a small nucleolar ribonucleoprotein particle (snoRNP) assembly factor facilitates condensin accessibility to the rDNA and induces nucleolar hyper-condensation. Interestingly, this increased compaction of the nucleolus interferes with the proper release of Cdc14 from this organelle. This observation provides an explanation for the delayed rDNA condensation in budding yeast, which is necessary to efficiently coordinate timely Cdc14 release and mitotic exit with nucleolar compaction and segregation.

GBPs Inhibit Motility of Shigella flexneri but Are Targeted for Degradation by the Bacterial Ubiquitin Ligase IpaH9.8.

  • Wandel MP
  • Cell Host Microbe
  • 2017 Oct 11

Literature context:


Abstract:

Interferon exposure boosts cell-autonomous immunity for more efficient pathogen control. But how interferon-enhanced immunity protects the cytosol against bacteria and how professionally cytosol-dwelling bacteria avoid clearance are insufficiently understood. Here we demonstrate that the interferon-induced GTPase family of guanylate-binding proteins (GBPs) coats Shigella flexneri in a hierarchical manner reliant on GBP1. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria but are antagonized by IpaH9.8, a bacterial ubiquitin ligase secreted into the host cytosol. IpaH9.8 ubiquitylates GBP1, GBP2, and GBP4 to cause the proteasome-dependent destruction of existing GBP coats. This ubiquitin coating of Shigella favors the pathogen as it liberates bacteria from GBP encapsulation to resume actin-mediated motility and cell-to-cell spread. We conclude that an important function of GBP recruitment to S. flexneri is to prevent the spread of infection to neighboring cells while IpaH9.8 helps bacterial propagation by counteracting GBP-dependent cell-autonomous immunity.

Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.

  • Li Y
  • Elife
  • 2017 Oct 24

Literature context:


Abstract:

Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlations between patient clinical phenotypes, electrophysiology in their RPEs, and the structure and function of BESTROPHIN1 mutant channels. Our results revealed that the disease-causing mechanism of BEST1 mutations is centered on the indispensable role of BESTROPHIN1 in mediating the long speculated Ca2+-dependent Cl- current in RPE, and demonstrate that the pathological potential of BEST1 mutations can be evaluated and predicted with our iPSC-based 'disease-in-a-dish' approach. Moreover, we demonstrated that patient RPE is rescuable with viral gene supplementation, providing a proof-of-concept for curing BEST1-associated diseases.

Funding information:
  • NEI NIH HHS - R00 EY025290()
  • NIGMS NIH HHS - P41 GM103403()

The Wound Microenvironment Reprograms Schwann Cells to Invasive Mesenchymal-like Cells to Drive Peripheral Nerve Regeneration.

  • Clements MP
  • Neuron
  • 2017 Sep 27

Literature context:


Abstract:

Schwann cell dedifferentiation from a myelinating to a progenitor-like cell underlies the remarkable ability of peripheral nerves to regenerate following injury. However, the molecular identity of the differentiated and dedifferentiated states in vivo has been elusive. Here, we profiled Schwann cells acutely purified from intact nerves and from the wound and distal regions of severed nerves. Our analysis reveals novel facets of the dedifferentiation response, including acquisition of mesenchymal traits and a Myc module. Furthermore, wound and distal dedifferentiated Schwann cells constitute different populations, with wound cells displaying increased mesenchymal character induced by localized TGFβ signaling. TGFβ promotes invasion and crosstalks with Eph signaling via N-cadherin to drive collective migration of the Schwann cells across the wound. Consistently, Tgfbr2 deletion in Schwann cells resulted in misdirected and delayed reinnervation. Thus, the wound microenvironment is a key determinant of Schwann cell identity, and it promotes nerve repair through integration of multiple concerted signals. VIDEO ABSTRACT.

RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo.

  • Wong HH
  • Neuron
  • 2017 Aug 16

Literature context:


Abstract:

Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo β-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal β-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits.

Neural Circuit-Specialized Astrocytes: Transcriptomic, Proteomic, Morphological, and Functional Evidence.

  • Chai H
  • Neuron
  • 2017 Aug 2

Literature context:


Abstract:

Astrocytes are ubiquitous in the brain and are widely held to be largely identical. However, this view has not been fully tested, and the possibility that astrocytes are neural circuit specialized remains largely unexplored. Here, we used multiple integrated approaches, including RNA sequencing (RNA-seq), mass spectrometry, electrophysiology, immunohistochemistry, serial block-face-scanning electron microscopy, morphological reconstructions, pharmacogenetics, and diffusible dye, calcium, and glutamate imaging, to directly compare adult striatal and hippocampal astrocytes under identical conditions. We found significant differences in electrophysiological properties, Ca2+ signaling, morphology, and astrocyte-synapse proximity between striatal and hippocampal astrocytes. Unbiased evaluation of actively translated RNA and proteomic data confirmed significant astrocyte diversity between hippocampal and striatal circuits. We thus report core astrocyte properties, reveal evidence for specialized astrocytes within neural circuits, and provide new, integrated database resources and approaches to explore astrocyte diversity and function throughout the adult brain. VIDEO ABSTRACT.

FoxO1 Promotes Mitophagy in the Podocytes of Diabetic Male Mice via the PINK1/Parkin Pathway.

  • Li W
  • Endocrinology
  • 2017 Jul 1

Literature context:


Abstract:

We recently showed that forkhead-box class O1 (FoxO1) activation protects against high glucose-induced injury by preventing mitochondrial dysfunction in the rat kidney cortex. In addition, FoxO1 has been reported to mediate putative kinase 1 (PINK1) transcription and promote autophagy in response to mitochondrial oxidative stress in murine cardiomyocytes. In this study, we ascertained whether overexpressing FoxO1 in the kidney cortex reverses preestablished diabetic nephropathy in animal models. The effect of FoxO1 on mitophagy signaling pathways was evaluated in mouse podocytes. In vivo experiments were performed in male KM mice. A mouse model of streptozotocin (STZ)-induced type 1 diabetes (T1D) was used, and lentiviral vectors were injected into the kidney cortex to overexpress FoxO1. A mouse podocyte cell line was treated with high concentrations of glucose and genetically modified using lentiviral vectors. We found aberrant mitochondrial morphology and reduced adenosine triphosphate production. These mitochondrial abnormalities were due to decreased mitophagy via reduced phosphatase/tensin homolog on chromosome 10-induced PINK1/Parkin-dependent signaling. FoxO1 upregulation and PINK1/Parkin pathway activation can individually restore injured podocytes in STZ-induced T1D mice. Our results link the antioxidative activity of FoxO1 with PINK1/Parkin-induced mitophagy, indicating a novel role of FoxO1 in diabetic nephropathy.

Funding information:
  • NCRR NIH HHS - P51RR165(United States)

Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy.

  • Joachim J
  • Curr. Biol.
  • 2017 Jul 24

Literature context:


Abstract:

Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.

Funding information:
  • NIGMS NIH HHS - R01 HD069647()
  • Wellcome Trust - R01 GM120776()

Deletion of RhoA in Progesterone Receptor-Expressing Cells Leads to Luteal Insufficiency and Infertility in Female Mice.

  • El Zowalaty AE
  • Endocrinology
  • 2017 Jul 1

Literature context:


Abstract:

Ras homolog gene family, member A (RhoA) is widely expressed throughout the female reproductive system. To assess its role in progesterone receptor-expressing cells, we generated RhoA conditional knockout mice RhoAd/d (RhoAf/f-Pgr-Cre+/-). RhoAd/d female mice had comparable mating activity, serum luteinizing hormone, prolactin, and estradiol levels and ovulation with control but were infertile with progesterone insufficiency, indicating impaired steroidogenesis in RhoAd/d corpus luteum (CL). RhoA was highly expressed in wild-type luteal cells and conditionally deleted in RhoAd/d CL. Gestation day 3.5 (D3.5) RhoAd/d ovaries had reduced numbers of CL, less defined corpus luteal cord formation, and disorganized CL collagen IV staining. RhoAd/d CL had lipid droplet and free cholesterol accumulation, indicating the availability of cholesterol for steroidogenesis, but disorganized β-actin and vimentin staining, indicating disrupted cytoskeleton integrity. Cytoskeleton is important for cytoplasmic cholesterol movement to mitochondria and for regulating mitochondria. Dramatically reduced expression of mitochondrial markers heat shock protein 60 (HSP60), voltage-dependent anion channel, and StAR was detected in RhoAd/d CL. StAR carries out the rate-limiting step of steroidogenesis. StAR messenger RNA expression was reduced in RU486-treated D3.5 wild-type CL and tended to be induced in progesterone-treated D3.5 RhoAd/d CL, with parallel changes of HSP60 expression. These data demonstrated the in vivo function of RhoA in CL luteal cell cytoskeleton integrity, cholesterol transport, StAR expression, and progesterone synthesis, and a positive feedback on StAR expression in CL by progesterone signaling. These findings provide insights into mechanisms of progesterone insufficiency.

Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function.

  • Sakamaki JI
  • Mol. Cell
  • 2017 May 18

Literature context:


Abstract:

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.

RPL10L Is Required for Male Meiotic Division by Compensating for RPL10 during Meiotic Sex Chromosome Inactivation in Mice.

  • Jiang L
  • Curr. Biol.
  • 2017 May 22

Literature context:


Abstract:

The mammalian sex chromosomes have undergone profound changes during their evolution from an ancestral pair of autosomes [1-4]. Specifically, the X chromosome has acquired a paradoxical sex-biased function by redistributing gene contents [5, 6] and has generated a disproportionately high number of retrogenes that are located on autosomes and exhibit male-biased expression patterns [6]. Several selection-based models have been proposed to explain this phenomenon, including a model of sexual antagonism driving X inactivation (SAXI) [6-8] and a compensatory mechanism based on meiotic sex chromosome inactivation (MSCI) [6, 8-11]. However, experimental evidence correlating the function of X-chromosome-derived autosomal retrogenes with evolutionary forces remains limited [12-17]. Here, we show that the deficiency of Rpl10l, a murine autosomal retrogene of Rpl10 with testis-specific expression, disturbs ribosome biogenesis in late-prophase spermatocytes and prohibits the transition from prophase into metaphase of the first meiotic division, resulting in male infertility. Rpl10l expression compensates for the lack of Rpl10, which exhibits a broad expression pattern but is subject to MSCI during spermatogenesis. Importantly, ectopic expression of RPL10L prevents the death of cultured RPL10-deficient somatic cells, and Rpl10l-promoter-driven transgenic expression of Rpl10 in spermatocytes restores spermatogenesis and fertility in Rpl10l-deficient mice. Our results demonstrate that Rpl10l plays an essential role during the meiotic stage of spermatogenesis by compensating for MSCI-mediated transcriptional silencing of Rpl10. These data provide direct evidence for the compensatory hypothesis and add novel insight into the evolution of X-chromosome-derived autosomal retrogenes and their role in male fertility.

Phosphorylation of NEUROG3 Links Endocrine Differentiation to the Cell Cycle in Pancreatic Progenitors.

  • Krentz NAJ
  • Dev. Cell
  • 2017 Apr 24

Literature context:


Abstract:

During pancreatic development, proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3), exit the cell cycle, and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183, which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle, NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G1/S cell-cycle checkpoint. Using models of mouse and human pancreas development, we show that lengthening of the G1 phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum, these studies demonstrate that progenitor cell-cycle G1 lengthening, through its actions on stabilization of NEUROG3, is an essential variable in normal endocrine cell genesis.

Funding information:
  • NIDDK NIH HHS - P30 DK063720()
  • NIDDK NIH HHS - R01 DK021344()
  • NIDDK NIH HHS - U01 DK089541()

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9.

  • van Tienen LM
  • Elife
  • 2017 Mar 15

Literature context:


Abstract:

Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing β-catenin via the Legless/BCL9 adaptor. We used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses.

Funding information:
  • Medical Research Council - MC_U105184273()
  • Medical Research Council - MC_U105192713()

Dysregulated NMDA-Receptor Signaling Inhibits Long-Term Depression in a Mouse Model of Fragile X Syndrome.

  • Toft AK
  • J. Neurosci.
  • 2016 Sep 21

Literature context:


Abstract:

Fragile X syndrome (FXS) is a neurodevelopmental disease. It is one of the leading monogenic causes of intellectual disability among boys with most also displaying autism spectrum disorder traits. Here we investigated the role of NMDA receptors on mGluR-dependent long-term depression (mGluR-LTD), a key biomarker in the disease, at four different developmental stages. First, we applied the mGluR agonist 3,5-dihydroxyphenylglycine in the absence or presence of the NMDAR blocker, APV, hereby unmasking the NMDAR component in this process. As expected, in the presence of APV, we found more LTD in the mouse KO than in WT. This, however, was only observed in the p30-60 age group. At all other age groups tested, mGluR-LTD was almost identical between KO and WT. Interestingly, at p60, in the absence of APV, no or very little LTD was found in KO that was completely restored by application of APV. This suggests that the underlying cause of the enhanced mGluR-LTD in KO (at p30) is caused by dysregulated NMDAR signaling. To investigate this further, we next used NMDAR-subunit-specific antagonists. Inhibition of GluN2B, but not GluN2A, blocked mGluR-LTD only in WT. This was in contrast in the KO where blocking GluN2B rescued mGluR-LTD, suggesting GluN2B-containing NMDARs in the KO are hyperactive. Thus, these findings suggest strong involvement of GluN2B-containing-NMDARs in the pathophysiology of FXS and highlight a potential path for treatment for the disease. SIGNIFICANCE STATEMENT: There is currently no cure for fragile X, although medications targeting specific FXS symptoms do exist. The FXS animal model, the Fmr1 knock-out mouse, has demonstrated an increased mGluR5-mediated long-term depression (LTD) leading to several clinical trials of mGluR5 inhibitors/modulators, yet all have failed. In addition, surprisingly little information exists about the possible role of other ion channels/receptors, including NMDA receptors (NMDAR), in mGluR-LTD. Here we focus on NMDARs and their regulation of mGluR-mediated LTD at different developmental stages using several different NMDAR blockers/antagonists. Our findings suggest dysregulated NMDARs in the pathophysiology of FXS leading to altered mGluR-mediated LTD. Together, these data will help to develop new drug candidates that could lead to reversal of the FXS phenotype.

Funding information:
  • Canadian Institutes of Health Research - (Canada)
  • HHMI - R01 NS092578(United States)

AgRP Neuron-Specific Deletion of Glucocorticoid Receptor Leads to Increased Energy Expenditure and Decreased Body Weight in Female Mice on a High-Fat Diet.

  • Shibata M
  • Endocrinology
  • 2016 Apr 2

Literature context:


Abstract:

Agouti-related protein (AgRP) expressed in the arcuate nucleus is a potent orexigenic neuropeptide, which increases food intake and reduces energy expenditure resulting in increases in body weight (BW). Glucocorticoids, key hormones that regulate energy balance, have been shown in rodents to regulate the expression of AgRP. In this study, we generated AgRP-specific glucocorticoid receptor (GR)-deficient (knockout [KO]) mice. Female and male KO mice on a high-fat diet (HFD) showed decreases in BW at the age of 6 weeks compared with wild-type mice, and the differences remained significant until 16 weeks old. The degree of resistance to diet-induced obesity was more robust in female than in male mice. On a chow diet, the female KO mice showed slightly but significantly attenuated weight gain compared with wild-type mice after 11 weeks, whereas there were no significant differences in BW in males between genotypes. Visceral fat pad mass was significantly decreased in female KO mice on HFD, whereas there were no significant differences in lean body mass between genotypes. Although food intake was similar between genotypes, oxygen consumption was significantly increased in female KO mice on HFD. In addition, the uncoupling protein-1 expression in the brown adipose tissues was increased in KO mice. These data demonstrate that the absence of GR signaling in AgRP neurons resulted in increases in energy expenditure accompanied by decreases in adiposity in mice fed HFD, indicating that GR signaling in AgRP neurons suppresses energy expenditure under HFD conditions.

Funding information:
  • NIDCD NIH HHS - R01DC012931(United States)
  • NIDDK NIH HHS - K01 DK081666(United States)

Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers.

  • Sharma V
  • Elife
  • 2016 Feb 19

Literature context:


Abstract:

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Diverse somatic mutation patterns and pathway alterations in human cancers" by Kan and colleagues published in Nature in 2010 (Kan et al., 2010). The experiments to be replicated are those reported in Figures 3D-F and 4C-F. Kan and colleagues utilized mismatch repair detection (MRD) technology to identify somatic mutations in primary human tumor samples and identified a previously uncharacterized arginine 243 to histidine (R243H) mutation in the G-protein α subunit GNAO1 in breast carcinoma tissue. In Figures 3D-F, Kan and colleagues demonstrated that stable expression of mutant GNAO1(R243D) conferred a significant growth advantage in human mammary epithelial cells, confirming the oncogenic potential of this mutation. Similarly, expression of variants with somatic mutations in MAP2K4, a JNK pathway kinase (shown in Figures 4C-E) resulted in a significant increase in anchorage-independent growth. Interestingly, these mutants exhibited reduced kinase activity compared to wild type MAP2K4, indicating these mutations impose a dominant-negative influence to promote growth (Figure 4F). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Funding information:
  • NEI NIH HHS - R01 EY026024(United States)

Reducing Adiposity in a Critical Developmental Window Has Lasting Benefits in Mice.

  • Lerea JS
  • Endocrinology
  • 2016 Feb 2

Literature context:


Abstract:

Although most adults can lose weight by dieting, a well-characterized compensatory decrease in energy expenditure promotes weight regain more than 90% of the time. Using mice with impaired hypothalamic leptin signaling as a model of early-onset hyperphagia and obesity, we explored whether this unfavorable response to weight loss could be circumvented by early intervention. Early-onset obesity was associated with impairments in the structure and function of brown adipose tissue mitochondria, which were ameliorated by weight loss at any age. Although decreased sympathetic tone in weight-reduced adults resulted in net reductions in brown adipose tissue thermogenesis and energy expenditure that promoted rapid weight regain, this was not the case when dietary interventions were initiated at weaning. Enhanced energy expenditure persisted even after mice were allowed to resume overeating, leading to lasting reductions in adiposity. These findings reveal a time window when dietary interventions can produce metabolic improvements that are stably maintained.

Funding information:
  • NEI NIH HHS - R01 EY020535(United States)

Osteocalcin Effect on Human β-Cells Mass and Function.

  • Sabek OM
  • Endocrinology
  • 2015 Sep 22

Literature context:


Abstract:

The osteoblast-specific hormone osteocalcin (OC) was found to regulate glucose metabolism, fat mass, and β-cell proliferation in mice. Here, we investigate the effect of decarboxylated OC (D-OC) on human β-cell function and mass in culture and in vivo using a Nonobese diabetic-severe combined immunodeficiency mouse model. We found that D-OC at dose ranges from 1.0 to 15 ng/mL significantly augmented insulin content and enhanced human β-cell proliferation of cultured human islets. This was paralleled by increased expression of sulfonylurea receptor protein; a marker of β-cell differentiation and a component of the insulin-secretory apparatus. Moreover, in a Nonobese diabetic-severe combined immunodeficiency mouse model, systemic administration of D-OC at 4.5-ng/h significantly augmented production of human insulin and C-peptide from the grafted human islets. Finally, histological staining of the human islet grafts showed that the improvement in the β-cell function was attributable to an increase in β-cell mass as a result of β-cell proliferation indicated by MKI67 staining together with the increased β-cell number and decreased α-cell number data obtained using laser scanning cytometry. Our data for the first time show D-OC-enhanced β-cell function in human islets and support future exploitation of D-OC-mediated β-cell regulation for developing useful clinical treatments for patients with diabetes.

Funding information:
  • NINDS NIH HHS - NS025044(United States)
  • Wellcome Trust - WT088357/Z/09/Z(United Kingdom)

Activins A and B Regulate Fate-Determining Gene Expression in Islet Cell Lines and Islet Cells From Male Mice.

  • Andrzejewski D
  • Endocrinology
  • 2015 Jul 20

Literature context:


Abstract:

TGFβ superfamily ligands, receptors, and second messengers, including activins A and B, have been identified in pancreatic islets and proposed to have important roles regulating development, proliferation, and function. We previously demonstrated that Fstl3 (an antagonist of activin activity) null mice have larger islets with β-cell hyperplasia and improved glucose tolerance and insulin sensitivity in the absence of altered β-cell proliferation. This suggested the hypothesis that increased activin signaling influences β-cell expansion by destabilizing the α-cell phenotype and promoting transdifferentiation to β-cells. We tested the first part of this hypothesis by treating α- and β-cell lines and sorted mouse islet cells with activin and related ligands. Treatment of the αTC1-6 α cell line with activins A or B suppressed critical α-cell gene expression, including Arx, glucagon, and MafB while also enhancing β-cell gene expression. In INS-1E β-cells, activin A treatment induced a significant increase in Pax4 (a fate determining β-cell gene) and insulin expression. In sorted primary islet cells, α-cell gene expression was again suppressed by activin treatment in α-cells, whereas Pax4 was enhanced in β-cells. Activin treatment in both cell lines and primary cells resulted in phosphorylated mothers against decapentaplegic-2 phosphorylation. Finally, treatment of αTC1-6 cells with activins A or B significantly inhibited proliferation. These results support the hypothesis that activin signaling destabilized the α-cell phenotype while promoting a β-cell fate. Moreover, these results support a model in which the β-cell expansion observed in Fstl3 null mice may be due, at least in part, to enhanced α- to β-cell transdifferentiation.

Funding information:
  • Canadian Institutes of Health Research - 43881(Canada)
  • NIDDK NIH HHS - R01DK069351(United States)

Defective regulation of the ubiquitin/proteasome system in the hypothalamus of obese male mice.

  • Ignacio-Souza LM
  • Endocrinology
  • 2014 Aug 19

Literature context:


Abstract:

In both human and experimental obesity, inflammatory damage to the hypothalamus plays an important role in the loss of the coordinated control of food intake and energy expenditure. Upon prolonged maintenance of increased body mass, the brain changes the defended set point of adiposity, and returning to normal weight becomes extremely difficult. Here we show that in prolonged but not in short-term obesity, the ubiquitin/proteasome system in the hypothalamus fails to maintain an adequate rate of protein recycling, leading to the accumulation of ubiquitinated proteins. This is accompanied by an increased colocalization of ubiquitin and p62 in the arcuate nucleus and reduced expression of autophagy markers in the hypothalamus. Genetic protection from obesity is accompanied by the normal regulation of the ubiquitin/proteasome system in the hypothalamus, whereas the inhibition of proteasome or p62 results in the acceleration of body mass gain in mice exposed for a short period to a high-fat diet. Thus, the defective regulation of the ubiquitin/proteasome system in the hypothalamus may be an important mechanism involved in the progression and autoperpetuation of obesity.

Funding information:
  • British Heart Foundation - FS/09/029/27902(United Kingdom)
  • NIDCD NIH HHS - P30 DC04657(United States)

Circadian dynamics of the cone-rod homeobox (CRX) transcription factor in the rat pineal gland and its role in regulation of arylalkylamine N-acetyltransferase (AANAT).

  • Rohde K
  • Endocrinology
  • 2014 Aug 19

Literature context:


Abstract:

The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

Funding information:
  • NINDS NIH HHS - R01NS038752(United States)

Autocrine and paracrine mechanisms of prostaglandin E₂ action on trophoblast/conceptus cells through the prostaglandin E₂ receptor (PTGER2) during implantation.

  • Waclawik A
  • Endocrinology
  • 2013 Oct 23

Literature context:


Abstract:

The conceptus and endometrium secrete large amounts of prostaglandin E₂ (PGE₂) into the porcine uterine lumen during the periimplantation period. We hypothesized that PGE₂ acts on conceptus/trophoblast cells through auto- and paracrine mechanisms. Real-time RT-PCR analysis revealed that PGE₂ receptor (PTGER)2 mRNA was 14-fold greater in conceptuses/trophoblasts on days 14-25 (implantation and early placentation period) vs preimplantation day 10-13 conceptuses (P < .05). Similarly, expression of PTGER2 protein increased during implantation. Conceptus expression of PTGER4 mRNA and protein did not differ on days 10-19. PGE₂ stimulated PTGER2 mRNA expression in day 15 trophoblast cells through PTGER2 receptor signaling. PGE₂ elevated aromatase expression and estradiol-17β secretion by trophoblast cells. Moreover, PGE₂ and the PTGER2 agonist, butaprost, increased the adhesive capacity of both human HTR-8/SVneo trophoblast and primary porcine trophoblast cells to extracellular matrix. This PGE₂-induced alteration in trophoblast cell adhesion to extracellular matrix was abolished by incubation of these cells with AH6809 (PTGER2 antagonist), ITGAVB3-directed tetrapeptide arg-gly-asp-ser or integrin ITGAVB3 antibody. PGE₂ stimulated adhesion of porcine trophoblast cells via the estrogen receptor and MEK/MAPK signaling pathway. PGE₂ induced phosphorylation of MAPK1/MAPK3 through PTGER2 and up-regulated expression of cell adhesion proteins such as focal adhesion kinase and intercellular adhesion molecule-1. Our study indicates that elevated PGE₂ in the periimplantation uterine lumen stimulates conceptus PTGER2 expression, which in turn promotes trophoblast adhesion via integrins, and synthesis and secretion of the porcine embryonic signal estradiol-17β. Moreover, the mechanism through which PGE₂ increases trophoblast adhesion is not species specific because it is PTGER2- and integrin-dependent in both porcine and human trophoblast cells.

Funding information:
  • NIGMS NIH HHS - 8P41GM103540(United States)