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Phospho-mTOR (Ser2448) Antibody

RRID:AB_330970

Antibody ID

AB_330970

Target Antigen

Phospho-mTOR (Ser2448) h, m, r, mk, mouse, non-human primate, rat, human

Proper Citation

(Cell Signaling Technology Cat# 2971, RRID:AB_330970)

Clonality

polyclonal antibody

Comments

Applications: W. Consolidation on 7/2016: AB_330971.

Host Organism

rabbit

Vendor

Cell Signaling Technology

Rescue of Learning and Memory Deficits in the Human Nonsyndromic Intellectual Disability Cereblon Knock-Out Mouse Model by Targeting the AMP-Activated Protein Kinase-mTORC1 Translational Pathway.

  • Bavley CC
  • J. Neurosci.
  • 2018 Mar 14

Literature context:


Abstract:

A homozygous nonsense mutation in the cereblon (CRBN) gene results in autosomal recessive, nonsyndromic intellectual disability that is devoid of other phenotypic features, suggesting a critical role of CRBN in mediating learning and memory. In this study, we demonstrate that adult male Crbn knock-out (CrbnKO) mice exhibit deficits in hippocampal-dependent learning and memory tasks that are recapitulated by focal knock-out of Crbn in the adult dorsal hippocampus, with no changes in social or repetitive behavior. Cellular studies identify deficits in long-term potentiation at Schaffer collateral CA1 synapses. We further show that Crbn is robustly expressed in the mouse hippocampus and CrbnKO mice exhibit hyperphosphorylated levels of AMPKα (Thr172). Examination of processes downstream of AMP-activated protein kinase (AMPK) finds that CrbnKO mice have a selective impairment in mediators of the mTORC1 translation initiation pathway in parallel with lower protein levels of postsynaptic density glutamatergic proteins and higher levels of excitatory presynaptic markers in the hippocampus with no change in markers of the unfolded protein response or autophagy pathways. Acute pharmacological inhibition of AMPK activity in adult CrbnKO mice rescues learning and memory deficits and normalizes hippocampal mTORC1 activity and postsynaptic glutamatergic proteins without altering excitatory presynaptic markers. Thus, this study identifies that loss of Crbn results in learning, memory, and synaptic defects as a consequence of exaggerated AMPK activity, inhibition of mTORC1 signaling, and decreased glutamatergic synaptic proteins. Thus, CrbnKO mice serve as an ideal model of intellectual disability to further explore molecular mechanisms of learning and memory.SIGNIFICANCE STATEMENT Intellectual disability (ID) is one of the most common neurodevelopmental disorders. The cereblon (CRBN) gene has been linked to autosomal recessive, nonsyndromic ID, characterized by an intelligence quotient between 50 and 70 but devoid of other phenotypic features, making cereblon an ideal protein for the study of the fundamental aspects of learning and memory. Here, using the cereblon knock-out mouse model, we show that cereblon deficiency disrupts learning, memory, and synaptic function via AMP-activated protein kinase hyperactivity, downregulation of mTORC1, and dysregulation of excitatory synapses, with no changes in social or repetitive behaviors, consistent with findings in the human population. This establishes the cereblon knock-out mouse as a model of pure ID without the confounding behavioral phenotypes associated with other current models of ID.

Funding information:
  • Medical Research Council - MC_U137761446(United Kingdom)
  • NIDA NIH HHS - R01 DA029122()

Systematic Functional Annotation of Somatic Mutations in Cancer.

  • Ng PK
  • Cancer Cell
  • 2018 Mar 12

Literature context:


Abstract:

The functional impact of the vast majority of cancer somatic mutations remains unknown, representing a critical knowledge gap for implementing precision oncology. Here, we report the development of a moderate-throughput functional genomic platform consisting of efficient mutant generation, sensitive viability assays using two growth factor-dependent cell models, and functional proteomic profiling of signaling effects for select aberrations. We apply the platform to annotate >1,000 genomic aberrations, including gene amplifications, point mutations, indels, and gene fusions, potentially doubling the number of driver mutations characterized in clinically actionable genes. Further, the platform is sufficiently sensitive to identify weak drivers. Our data are accessible through a user-friendly, public data portal. Our study will facilitate biomarker discovery, prediction algorithm improvement, and drug development.

Funding information:
  • NCI NIH HHS - P30 CA016672()
  • NHLBI NIH HHS - HL-090775(United States)

Glucocorticoid Receptor Signaling Impairs Protein Turnover Regulation in Hypoxia-Induced Muscle Atrophy in Male Mice.

  • de Theije CC
  • Endocrinology
  • 2018 Jan 1

Literature context:


Abstract:

Hypoxemia may contribute to muscle wasting in conditions such as chronic obstructive pulmonary disease. Muscle wasting develops when muscle proteolysis exceeds protein synthesis. Hypoxia induces skeletal muscle atrophy in mice, which can in part be attributed to reduced food intake. We hypothesized that hypoxia elevates circulating corticosterone concentrations by reduced food intake and enhances glucocorticoid receptor (GR) signaling in muscle, which causes elevated protein degradation signaling and dysregulates protein synthesis signaling during hypoxia-induced muscle atrophy. Muscle-specific GR knockout and control mice were subjected to normoxia, normobaric hypoxia (8% oxygen), or pair-feeding to the hypoxia group for 4 days. Plasma corticosterone and muscle GR signaling increased after hypoxia and pair-feeding. GR deficiency prevented muscle atrophy by pair-feeding but not by hypoxia. GR deficiency differentially affected activation of ubiquitin 26S-proteasome and autophagy proteolytic systems by pair-feeding and hypoxia. Reduced food intake suppressed mammalian target of rapamycin complex 1 (mTORC1) activity under normoxic but not hypoxic conditions, and this retained mTORC1 activity was mediated by GR. We conclude that GR signaling is required for muscle atrophy and increased expression of proteolysis-associated genes induced by decreased food intake under normoxic conditions. Under hypoxic conditions, muscle atrophy and elevated gene expression of the ubiquitin proteasomal system-associated E3 ligases Murf1 and Atrogin-1 are mostly independent of GR signaling. Furthermore, impaired inhibition of mTORC1 activity is GR-dependent in hypoxia-induced muscle atrophy.

MicroRNA501-5p induces p53 proteasome degradation through the activation of the mTOR/MDM2 pathway in ADPKD cells.

  • de Stephanis L
  • J. Cell. Physiol.
  • 2018 Jan 13

Literature context:


Abstract:

Cell proliferation and apoptosis are typical hallmarks of autosomal dominant polycystic kidney disease (ADPKD) and cause the development of kidney cysts that lead to end-stage renal disease (ESRD). Many factors, impaired by polycystin complex loss of function, may promote these biological processes, including cAMP, mTOR, and EGFR signaling pathways. In addition, microRNAs (miRs) may also regulate the ADPKD related signaling network and their dysregulation contributes to disease progression. However, the role of miRs in ADPKD pathogenesis has not been fully understood, but also the function of p53 is quite obscure, especially its regulatory contribution on cell proliferation and apoptosis. Here, we describe for the first time that miR501-5p, upregulated in ADPKD cells and tissues, induces the activation of mTOR kinase by PTEN and TSC1 gene repression. The increased activity of mTOR kinase enhances the expression of E3 ubiquitin ligase MDM2 that in turn promotes p53 ubiquitination, leading to its degradation by proteasome machinery in a network involving p70S6K. Moreover, the overexpression of miR501-5p stimulates cell proliferation in kidney cells by the inhibition of p53 function in a mechanism driven by mTOR signaling. In fact, the downregulation of this miR as well as the pharmacological treatment with proteasome and mTOR inhibitors in ADPKD cells reduces cell growth by the activation of apoptosis. Consequently, the stimulation of cell death in ADPKD cells may occur through the inhibition of mTOR/MDM2 signaling and the restoring of p53 function. The data presented here confirm that the impaired mTOR signaling plays an important role in ADPKD.

Funding information:
  • NIAID NIH HHS - 1R01AI075077-01A1(United States)

Genetic Removal of eIF2α Kinase PERK in Mice Enables Hippocampal L-LTP Independent of mTORC1 Activity.

  • Zimmermann HR
  • J. Neurochem.
  • 2018 Jan 16

Literature context:


Abstract:

Characterization of the molecular signaling pathways underlying protein synthesis-dependent forms of synaptic plasticity, such as late long-term potentiation (L-LTP), can provide insights not only into memory expression/maintenance under physiological conditions but also potential mechanisms associated with the pathogenesis of memory disorders. Here, we report in mice that L-LTP failure induced by the mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) inhibitor rapamycin is reversed by brain-specific genetic deletion of PKR-like ER kinase, PERK (PERK KO), a kinase for eukaryotic initiation factor 2α (eIF2α). In contrast, genetic removal of general control non-derepressible-2, GCN2 (GCN2 KO), another eIF2α kinase, or treatment of hippocampal slices with the PERK inhibitor GSK2606414, does not rescue rapamycin-induced L-LTP failure, suggesting mechanisms independent of eIF2α phosphorylation. Moreover, we demonstrate that phosphorylation of eukaryotic elongation factor 2 (eEF2) is significantly decreased in PERK KO mice but unaltered in GCN2 KO mice or slices treated with the PERK inhibitor. Reduction of eEF2 phosphorylation results in increased general protein synthesis, and thus could contribute to the mTORC1-independent L-LTP in PERK KO mice. We further performed experiments on mutant mice with genetic removal of eEF2K (eEF2K KO), the only known kinase for eEF2, and found that L-LTP in eEF2K KO mice is insensitive to rapamycin. These data, for the first time, connect reduction of PERK activity with the regulation of translation elongation in enabling L-LTP independent of mTORC1. Thus, our findings indicate previously unrecognized levels of complexity in the regulation of protein synthesis-dependent synaptic plasticity. This article is protected by copyright. All rights reserved.

Funding information:
  • NIA NIH HHS - F31 AG054113()
  • NIA NIH HHS - K99 AG044469()
  • NIA NIH HHS - R00 AG044469()
  • NIA NIH HHS - R01 AG055581()
  • NIA NIH HHS - R01 AG056622()
  • NIGMS NIH HHS - T32 GM007739(United States)

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

  • Essig K
  • Immunity
  • 2017 Dec 19

Literature context:


Abstract:

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Funding information:
  • NHLBI NIH HHS - R37 HL036982(United States)

A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice.

  • Roberts MN
  • Cell Metab.
  • 2017 Sep 5

Literature context:


Abstract:

Calorie restriction, without malnutrition, has been shown to increase lifespan and is associated with a shift away from glycolysis toward beta-oxidation. The objective of this study was to mimic this metabolic shift using low-carbohydrate diets and to determine the influence of these diets on longevity and healthspan in mice. C57BL/6 mice were assigned to a ketogenic, low-carbohydrate, or control diet at 12 months of age and were either allowed to live their natural lifespan or tested for physiological function after 1 or 14 months of dietary intervention. The ketogenic diet (KD) significantly increased median lifespan and survival compared to controls. In aged mice, only those consuming a KD displayed preservation of physiological function. The KD increased protein acetylation levels and regulated mTORC1 signaling in a tissue-dependent manner. This study demonstrates that a KD extends longevity and healthspan in mice.

Funding information:
  • NIA NIH HHS - P01 AG025532()
  • NIDDK NIH HHS - U24 DK092993()

Pimozide reduces toxic forms of tau in TauC3 mice via 5' adenosine monophosphate-activated protein kinase-mediated autophagy.

  • Kim YD
  • J. Neurochem.
  • 2017 Sep 20

Literature context:


Abstract:

In neurodegenerative diseases like Alzheimer's disease (AD), tau is hyperphosphorylated and forms aggregates and neurofibrillary tangles in affected neurons. Autophagy is critical to clear the aggregates of disease-associated proteins and is often altered in patients and animal models of AD. Because mechanistic target of rapamycin (mTOR) negatively regulates autophagy and is hyperactive in the brains of patients with AD, mTOR is an attractive therapeutic target for AD. However, pharmacological strategies to increase autophagy by targeting mTOR inhibition cause various side effects. Therefore, autophagy activation mediated by non-mTOR pathways is a new option for autophagy-based AD therapy. Here, we report that pimozide activates autophagy to rescue tau pathology in an AD model. Pimozide increased autophagic flux through the activation of the AMPK-Unc-51 like autophagy activating kinase 1 (ULK1) axis, but not of mTOR, in neuronal cells, and this function was independent of dopamine D2 receptor inhibition. Pimozide reduced levels of abnormally phosphorylated tau aggregates in neuronal cells. Further, daily intraperitoneal (i.p.) treatment of pimozide led to a recovery from memory deficits of TauC3 mice expressing a caspase-cleaved form of tau. In the brains of these mice, we found increased phosphorylation of AMPK1 and ULK1, and reduced levels of the soluble oligomers and NP40-insoluble aggregates of abnormally phosphorylated tau. Together, these results suggest that pimozide rescues memory impairments in TauC3 mice and reduces tau aggregates by increasing autophagic flux through the mTOR-independent AMPK-ULK1 axis.

TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease.

  • Ulland TK
  • Cell
  • 2017 Aug 10

Literature context:


Abstract:

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

Funding information:
  • NCI NIH HHS - T32 CA009547()
  • NIA NIH HHS - P01 AG003991()
  • NIA NIH HHS - P01 AG026276()
  • NIA NIH HHS - P50 AG005681()
  • NIA NIH HHS - RF1 AG051485()
  • NIDDK NIH HHS - R01 DK058177()

Clk1-regulated aerobic glycolysis is involved in glioma chemoresistance.

  • Zhang L
  • J. Neurochem.
  • 2017 Aug 5

Literature context:


Abstract:

Chemoresistance remains a major challenge for the treatment of glioma. In this study, we investigated the role of Clock 1 (Clk1), which encodes an enzyme that is necessary for ubiquinone biosynthesis in glioma chemoresistance in vitro. The results showed that Clk1 was highly expressed in GL261 mouse glioma cells which were most sensitive to 1,3Bis (2-chloroethyl) 1 nitrosourea (BCNU) while was low expressed in BCNU resistant cells such as glioma cancer stem cells, T98G, U87MG and U251 glioma cells. Knockdown of Clk1 in GL261 glioma cells significantly reduced BCNU- or cisplatin-induced cell apoptosis, whereas the proliferative activity and the expression of multidrug resistance-related genes including MDR1, O6-methylguanine-DNA methyltransferase, and GSTP1 were not changed. When Clk1 was re-expressed in Clk1 knockdown GL261 glioma cells, the BCNU sensitivity was restored. The mechanistic study revealed that knockdown of Clk1 in GL261 glioma cells increased aerobic glycolysis including high glucose consumption, lactate production, and up-regulation of glycolysis-associated genes. Inhibition of glycolysis can reverse the chemoresistance elicited by Clk1 knockdown in GL261 cells. Moreover, knockdown of Clk1 induced HIF-1α expression in GL261 glioma cells which was found to be mediated by AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling pathway. Both metformin and rapamycin reversed the chemoresistance of Clk1 knockdown GL261 glioma cells. Over-expression of Clk1 significantly increased the sensitivity of T98G or U251 human glioblastoma cells to BCNU which was accompanied by decreased lactate secretion, decreased expression of HIF-1α, AMPK activation, and inhibition of mTOR pathway. Inhibition of glycolysis or activation of AMPK did not alter Clk1 expression in variant glioma cell lines suggesting that aerobic glycolysis is not an upstream event of Clk1 expression in glioma cells. Taken together, our results revealed, for the first time, that mitochondrial Clk1 regulated chemoresistance in glioma cells through AMPK/mTOR/HIF-1α mediated glycolysis pathway.

Hypothalamic Regulation of Liver and Muscle Nutrient Partitioning by Brain-Specific Carnitine Palmitoyltransferase 1C in Male Mice.

  • Pozo M
  • Endocrinology
  • 2017 Jul 1

Literature context:


Abstract:

Carnitine palmitoyltransferase (CPT) 1C, a brain-specific protein localized in the endoplasmic reticulum of neurons, is expressed in almost all brain regions. Based on global knockout (KO) models, CPT1C has demonstrated relevance in hippocampus-dependent spatial learning and in hypothalamic regulation of energy balance. Specifically, it has been shown that CPT1C is protective against high-fat diet-induced obesity (DIO), and that CPT1C KO mice show reduced peripheral fatty acid oxidation (FAO) during both fasting and DIO. However, the mechanisms mediating CPT1C-dependent regulation of energy homeostasis remain unclear. Here, we focus on the mechanistic understanding of hypothalamic CPT1C on the regulation of fuel selection in liver and muscle of male mice during energy deprivation situations, such as fasting. In CPT1C-deficient mice, modulation of the main hypothalamic energy sensors (5' adenosine monophosphate-activated protein kinase, Sirtuin 1, and mammalian target of rapamycin) was impaired and plasma catecholamine levels were decreased. Consequently, CPT1C-deficient mice presented defective fasting-induced FAO in liver, leading to higher triacylglycerol accumulation and lower glycogen levels. Moreover, muscle pyruvate dehydrogenase activity was increased, which was indicative of glycolysis enhancement. The respiratory quotient did not decrease in CPT1C KO mice after 48 hours of fasting, confirming a defective switch on fuel substrate selection under hypoglycemia. Phenotype reversion studies identified the mediobasal hypothalamus (MBH) as the main area mediating CPT1C effects on fuel selection. Overall, our data demonstrate that CPT1C in the MBH is necessary for proper hypothalamic sensing of a negative energy balance and fuel partitioning in liver and muscle.

Funding information:
  • NICHD NIH HHS - U54 HD086984(United States)
  • NIMH NIH HHS - MH059490(United States)

Chronic Hyperinsulinemia Increases Myoblast Proliferation in Fetal Sheep Skeletal Muscle.

  • Brown LD
  • Endocrinology
  • 2017 Jun 5

Literature context:


Abstract:

Insulin is an important fetal growth factor. However, chronic experimental hyperinsulinemia in the fetus fails to accelerate linear and lean mass growth beyond normal rates. Mechanisms preventing accelerated lean mass accretion during hyperinsulinemia are unknown. To address potential mechanisms, late-gestation fetal sheep were infused with iv insulin and glucose to produce euglycemic hyperinsulinemia (INS) or saline for 7-9 days. Fetal substrate uptake and protein metabolic rates were measured. INS fetuses had 1.5-fold higher insulin concentrations (P < .0001) and equivalent glucose concentrations. INS fetuses had 20% more Pax7(+) nuclei in the biceps femoris, which indicates the potential for hyperinsulinemia to increase the number of myoblasts within late-gestation fetal skeletal muscle. Additionally, the percentage of Pax7(+) myoblasts that expressed Ki-67 was 1.3-fold higher and expression of myogenic regulatory factors was 50% lower in INS fetuses (MYF5 and MYOG [myogenin], P < .005), which indicates a shift toward myoblast proliferation over differentiation. There were no differences for fetal body, organ, or muscle weights, although INS placentas weighed 28% less (P < .05). Protein synthesis and accretion rates did not change in INS fetuses, nor did fiber muscle size. Essential amino acid concentrations were lower in the INS group (P < .05) except for tryptophan. Umbilical blood flow, net total amino acids, and O2 uptakes rates did not differ between groups. Arterial O2 content was 33% lower (P < .005) and norepinephrine was 100% higher in the INS fetuses (P < .01), all of which are factors that may counteract fetal protein accretion during hyperinsulinemia despite an increase in myoblast proliferation.

Funding information:
  • NIGMS NIH HHS - T32 GM007377(United States)

Developmental Programming: Insulin Sensitizer Prevents the GnRH-Stimulated LH Hypersecretion in a Sheep Model of PCOS.

  • Cardoso RC
  • Endocrinology
  • 2017 May 26

Literature context:


Abstract:

Prenatal testosterone (T) treatment recapitulates the reproductive and metabolic phenotypes of polycystic ovary syndrome in female sheep. At the neuroendocrine level, prenatal T treatment results in disrupted steroid feedback on gonadotropin release, increased pituitary sensitivity to GnRH, and subsequent LH hypersecretion. Because prenatal T-treated sheep manifest functional hyperandrogenism and hyperinsulinemia, gonadal steroids and/or insulin may play a role in programming and/or maintaining these neuroendocrine defects. Here, we investigated the effects of prenatal and postnatal treatments with an androgen antagonist (flutamide [F]) or an insulin sensitizer (rosiglitazone [R]) on GnRH-stimulated LH secretion in prenatal T-treated sheep. As expected, prenatal T treatment increased the pituitary responsiveness to GnRH leading to LH hypersecretion. Neither prenatal interventions nor postnatal F treatment normalized the GnRH-stimulated LH secretion. Conversely, postnatal R treatment completely normalized the GnRH-stimulated LH secretion. At the tissue level, gestational T increased pituitary LHβ, androgen receptor, and insulin receptor-β, whereas it reduced estrogen receptor (ER)α protein levels. Although postnatal F normalized pituitary androgen receptor and insulin receptor-β, it failed to prevent an increase in LHβ expression. Contrarily, postnatal R treatment restored ERα and partially normalized LHβ pituitary levels. Immunohistochemical findings confirmed changes in pituitary ERα expression to be specific to gonadotropes. In conclusion, these findings indicate that increased pituitary responsiveness to GnRH in prenatal T-treated sheep is likely a function of reduced peripheral insulin sensitivity. Moreover, results suggest that restoration of ERα levels in the pituitary may be one mechanism by which R prevents GnRH-stimulated LH hypersecretion in this sheep model of polycystic ovary syndrome-like phenotype.

Funding information:
  • NIAMS NIH HHS - AR061933(United States)

Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling.

  • Hill SM
  • Cell Syst
  • 2017 Jan 25

Literature context:


Abstract:

Signaling networks downstream of receptor tyrosine kinases are among the most extensively studied biological networks, but new approaches are needed to elucidate causal relationships between network components and understand how such relationships are influenced by biological context and disease. Here, we investigate the context specificity of signaling networks within a causal conceptual framework using reverse-phase protein array time-course assays and network analysis approaches. We focus on a well-defined set of signaling proteins profiled under inhibition with five kinase inhibitors in 32 contexts: four breast cancer cell lines (MCF7, UACC812, BT20, and BT549) under eight stimulus conditions. The data, spanning multiple pathways and comprising ∼70,000 phosphoprotein and ∼260,000 protein measurements, provide a wealth of testable, context-specific hypotheses, several of which we experimentally validate. Furthermore, the data provide a unique resource for computational methods development, permitting empirical assessment of causal network learning in a complex, mammalian setting.

Funding information:
  • Medical Research Council - MC_UP_0801/1()
  • Medical Research Council - MC_UP_1302/3()
  • NCI NIH HHS - P30 CA016672()
  • NCI NIH HHS - U54 CA112970()

IGF-I and IGFBP-2 Stimulate AMPK Activation and Autophagy, Which Are Required for Osteoblast Differentiation.

  • Xi G
  • Endocrinology
  • 2016 Jan 31

Literature context:


Abstract:

IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3-6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.

Funding information:
  • NIDA NIH HHS - 5R01DA023210-02(United States)

Role of VGF-derived carboxy-terminal peptides in energy balance and reproduction: analysis of "humanized" knockin mice expressing full-length or truncated VGF.

  • Sadahiro M
  • Endocrinology
  • 2015 May 18

Literature context:


Abstract:

Targeted deletion of VGF, a secreted neuronal and endocrine peptide precursor, produces lean, hypermetabolic, and infertile mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes. Previous studies suggest that VGF controls energy expenditure (EE), fat storage, and lipolysis, whereas VGF C-terminal peptides also regulate reproductive behavior and glucose homeostasis. To assess the functional equivalence of human VGF(1-615) (hVGF) and mouse VGF(1-617) (mVGF), and to elucidate the function of the VGF C-terminal region in the regulation of energy balance and susceptibility to obesity, we generated humanized VGF knockin mouse models expressing full-length hVGF or a C-terminally deleted human VGF(1-524) (hSNP), encoded by a single nucleotide polymorphism (rs35400704). We show that homozygous male and female hVGF and hSNP mice are fertile. hVGF female mice had significantly increased body weight compared with wild-type mice, whereas hSNP mice have reduced adiposity, increased activity- and nonactivity-related EE, and improved glucose tolerance, indicating that VGF C-terminal peptides are not required for reproductive function, but 1 or more specific VGF C-terminal peptides are likely to be critical regulators of EE. Taken together, our results suggest that human and mouse VGF proteins are largely functionally conserved but that species-specific differences in VGF peptide function, perhaps a result of known differences in receptor binding affinity, likely alter the metabolic phenotype of hVGF compared with mVGF mice, and in hSNP mice in which several C-terminal VGF peptides are ablated, result in significantly increased activity- and nonactivity-related EE.

Funding information:
  • NINDS NIH HHS - N01NS02331(United States)

Role of androgens in sex differences in cardiac damage during myocardial infarction.

  • Le TY
  • Endocrinology
  • 2014 Feb 22

Literature context:


Abstract:

Age-specific incidence of ischemic heart disease in men is higher than in women, although women die more frequently without previous symptoms; the molecular mechanism(s) are poorly understood. Most studies focus on protection by estrogen, with less attention on androgen receptor-mediated androgen actions. Our aim was to determine the role of androgens in the sex differences in cardiac damage during myocardial infarction. Mature age-matched male and female Sprague Dawley rats, intact or surgically gonadectomized (Gx), received testosterone (T) or 17β-estradiol (E2) via subdermal SILASTIC (Dow Corning Corp.) implants; a subset of male rats received dihydrotestosterone. After 21 days, animals were anesthetized, and hearts were excised and subjected to ex vivo regional ischemia-reperfusion (I-R). Hearts from intact males had larger infarcts than those from females following I-R; Gx produced the opposite effect, confirming a role for sex steroids. In Gx males, androgens (dihydrotestosterone, T) and E2 aggravated I-R-induced cardiac damage, whereas in Gx females, T had no effect and E2 reduced infarct area. Increased circulating T levels up-regulated androgen receptor and receptor for advanced glycation end products, which resulted in enhanced apoptosis aggravating cardiac damage in both males and females. In conclusion, our study demonstrates, for the first time, that sex steroids regulate autophagy during myocardial infarction and shows that a novel mechanism of action for androgens during I-R is down-regulation of antiapoptotic protein Bcl-xL (B cell lymphoma-extra large), a key controller for cross talk between autophagy and apoptosis, shifting the balance toward apoptosis and leading to aggravated cardiac damage.

Funding information:
  • Intramural NIH HHS - (United States)
  • NIAID NIH HHS - R21 AI102187(United States)

Regulation of LH receptor mRNA binding protein by miR-122 in rat ovaries.

  • Menon B
  • Endocrinology
  • 2013 Dec 25

Literature context:


Abstract:

LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5'-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.

Funding information:
  • NEI NIH HHS - EY019051(United States)

Maternal dietary restriction during the periconceptional period in normal-weight or obese ewes results in adrenocortical hypertrophy, an up-regulation of the JAK/STAT and down-regulation of the IGF1R signaling pathways in the adrenal of the postnatal lamb.

  • Zhang S
  • Endocrinology
  • 2013 Dec 25

Literature context:


Abstract:

Maternal dietary restriction during the periconceptional period results in an increase in adrenal growth and in the cortisol stress response in the offspring. The intraadrenal mechanisms that result in the programming of these changes are not clear. Activation of the IGF and the signal transducer and activator of transcription (STAT)/suppressors of cytokine signaling (SOCS) pathways regulate adrenal growth. We have used an embryo transfer model in sheep to investigate the impact of exposure to either dietary restriction in normal or obese mothers or to maternal obesity during the periconceptional period on adrenal growth and function in the offspring. We assessed the adrenal abundance of key signaling molecules in the IGF-I and Janus kinase/STAT/SOCS pathways including IGF-I receptor, IGF-II receptor, Akt, mammalian target of rapamycin, ribosomal protein S6, eukaryotic translation initiation factor 4E-binding protein 1, eukaryotic translation initiation factor 4E, STAT1, STAT3, STAT5, SOCS1, and SOCS3 in female and male postnatal lambs. Maternal dietary restriction in the periconceptional period resulted in the hypertrophy of the adrenocortical cells in the zona fasciculata-reticularis and an up-regulation in STAT1, phospho-STAT1, and phospho-STAT3 (Ser727) abundance and a down-regulation in IGF-I receptor, Akt, and phospho-Akt abundance in the adrenal cortex of the postnatal lamb. These studies highlight that weight loss around the time of conception, independent of the starting maternal body weight, results in the activation of the adrenal Janus kinase/STAT pathway and adrenocortical hypertrophy. Thus, signals of adversity around the time of conception have a long-term impact on the mechanisms that regulate adrenocortical growth.

Funding information:
  • NHLBI NIH HHS - R01 HL-085848(United States)
  • NINDS NIH HHS - R01 NS070300(United States)