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GAPDH antibody

RRID:AB_307275

Antibody ID

AB_307275

Target Antigen

GAPDH antibody human, mouse, rat, chicken, saccharomyces cerevisiae, schizosaccharomyces pombe, xenopus laevis, xenopus/amphibian, human, chicken/bird, mouse, rat, yeast/fungi

Proper Citation

(Abcam Cat# ab9485, RRID:AB_307275)

Clonality

polyclonal antibody

Comments

validation status unknown, seller recommendations provided in 2012: ELISA, Flow Cyt, ICC/IF, IHC-Fr, IP, WB; Immunofluorescence; Other; Flow Cytometry; ELISA; Immunohistochemistry; Immunohistochemistry - frozen; Western Blot; Immunocytochemistry; Immunoprecipitation

Host Organism

rabbit

Vendor

Abcam

A Designed Peptide Targets Two Types of Modifications of p53 with Anti-cancer Activity.

  • Liang L
  • Cell Chem Biol
  • 2018 Jun 21

Literature context:


Abstract:

Many cancer-related proteins are controlled by composite post-translational modifications (PTMs), but prevalent strategies only target one type of modification. Here we describe a designed peptide that controls two types of modifications of the p53 tumor suppressor, based on the discovery of a protein complex that suppresses p53 (suppresome). We found that Morn3, a cancer-testis antigen, recruits different PTM enzymes, such as sirtuin deacetylase and ubiquitin ligase, to confer composite modifications on p53. The molecular functions of Morn3 were validated through in vivo assays and chemico-biological intervention. A rationally designed Morn3-targeting peptide (Morncide) successfully activated p53 and suppressed tumor growth. These findings shed light on the regulation of protein PTMs and present a strategy for targeting two modifications with one molecule.

Funding information:
  • NCI NIH HHS - K08 CA102545(United States)

Phosphatidylinositol-5-Phosphate 4-Kinases Regulate Cellular Lipid Metabolism By Facilitating Autophagy.

  • Lundquist MR
  • Mol. Cell
  • 2018 May 3

Literature context:


Abstract:

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P2) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P2, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a-/-Pip4k2b-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P2 synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.

Funding information:
  • NCI NIH HHS - R35 CA197588()
  • NCI NIH HHS - U54 CA210184()
  • NCRR NIH HHS - UL1RR024128(United States)
  • NIGMS NIH HHS - R01 GM041890()

GABAA and GABAB receptor subunit localization on neurochemically identified neurons of the human subthalamic nucleus.

  • Wu XH
  • J. Comp. Neurol.
  • 2018 Apr 1

Literature context:


Abstract:

The subthalamic nucleus (STN) is a critical excitatory signaling center within the basal ganglia circuitry. The activity of subthalamic neurons is tightly controlled by upstream inhibitory signaling centers in the basal ganglia. In this study, we used immunohistochemical techniques to firstly, visualize and quantify the STN neurochemical organization based on neuronal markers including parvalbumin (PV), calretinin (CR), SMI-32, and GAD65/67 . Secondly, we characterized the detailed regional, cellular and subcellular expression of GABAA (α1 , α2 , α3 , β2/3 , and γ2 ) and GABAB (R1 and R2) receptor subunits within the normal human STN. Overall, we found seven neurochemically distinct populations of principal neurons in the human STN. The three main populations detected were: (a) triple-labeled PV+ /CR+ /SMI32+ ; (b) double-labeled PV+ /CR+ ; and (c) single-labeled CR+ neurons. Subthalamic principal neurons were found to express GABAA receptor subunits α1 , α3 , β2/3 , γ2 , and GABAB receptor subunits R1 and R2. However, no expression of GABAA receptor α2 subunit was detected. We also found a trend of increasing regional staining intensity for all positive GABAA receptor subunits from the dorsolateral pole to ventromedial extremities. The GAD+ interneurons showed relatively low expression of GABAA receptor subunits. These results provide the morphological basis of GABAergic transmission within the normal human subthalamic nucleus and evidence of GABA innervation through both GABAA and GABAB receptors on subthalamic principal neurons.

Funding information:
  • NIAID NIH HHS - T32AI007061(United States)

N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H2S and Sulfane Sulfur Production.

  • Ezeriņa D
  • Cell Chem Biol
  • 2018 Apr 19

Literature context:


Abstract:

The cysteine prodrug N-acetyl cysteine (NAC) is widely used as a pharmacological antioxidant and cytoprotectant. It has been reported to lower endogenous oxidant levels and to protect cells against a wide range of pro-oxidative insults. As NAC itself is a poor scavenger of oxidants, the molecular mechanisms behind the antioxidative effects of NAC have remained uncertain. Here we show that NAC-derived cysteine is desulfurated to generate hydrogen sulfide, which in turn is oxidized to sulfane sulfur species, predominantly within mitochondria. We provide evidence suggesting the possibility that sulfane sulfur species produced by 3-mercaptopyruvate sulfurtransferase and sulfide:quinone oxidoreductase are the actual mediators of the immediate antioxidative and cytoprotective effects provided by NAC.

Funding information:
  • NIGMS NIH HHS - R01 GM056264-07(United States)

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation.

  • Simões AP
  • Cell Death Dis
  • 2018 Feb 20

Literature context:


Abstract:

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Funding information:
  • NCRR NIH HHS - 5P20-RR021940(United States)

Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System.

  • Weng YL
  • Neuron
  • 2018 Jan 17

Literature context:


Abstract:

N6-methyladenosine (m6A) affects multiple aspects of mRNA metabolism and regulates developmental transitions by promoting mRNA decay. Little is known about the role of m6A in the adult mammalian nervous system. Here we report that sciatic nerve lesion elevates levels of m6A-tagged transcripts encoding many regeneration-associated genes and protein translation machinery components in the adult mouse dorsal root ganglion (DRG). Single-base resolution m6A-CLIP mapping further reveals a dynamic m6A landscape in the adult DRG upon injury. Loss of either m6A methyltransferase complex component Mettl14 or m6A-binding protein Ythdf1 globally attenuates injury-induced protein translation in adult DRGs and reduces functional axon regeneration in the peripheral nervous system in vivo. Furthermore, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult central nervous system is attenuated upon Mettl14 knockdown. Our study reveals a critical epitranscriptomic mechanism in promoting injury-induced protein synthesis and axon regeneration in the adult mammalian nervous system.

Funding information:
  • NCI NIH HHS - U01 CA84243(United States)
  • NHGRI NIH HHS - RM1 HG008935()
  • NINDS NIH HHS - P01 NS097206()
  • NINDS NIH HHS - R35 NS097370()

Glucocorticoids Suppress the Browning of Adipose Tissue via miR-19b in Male Mice.

  • Lv YF
  • Endocrinology
  • 2018 Jan 1

Literature context:


Abstract:

Physiological levels of glucocorticoids (GCs) are required for proper metabolic control, and excessive GC action has been linked to a variety of pandemic metabolic diseases. MicroRNA (miRNA)-19b plays a critical role in the pathogenesis of GC-induced metabolic diseases. This study explored the potential of miRNA-based therapeutics targeting adipose tissue. Our results showed that overexpressed miR-19b in stromal vascular fraction (SVF) cells derived from subcutaneous adipose tissue had the same effects as dexamethasone (DEX) treatment on the inhibition of adipose browning and oxygen consumption rate. The inhibition of miR-19b blocked DEX-mediated suppression of the expression of browning marker genes as well as the oxygen consumption rate in differentiated SVF cells derived from subcutaneous and brown adipose tissue. Overexpressed miR-19b in SVF cells derived from brown adipose tissue had the same effects as DEX treatment on the inhibition of brown adipose differentiation and energy expenditure. Glucocorticoids transcriptionally regulate the expression of miR-19b via a GC receptor-mediated direct DNA binding mechanism. This study confirmed that miR-19b is an essential target for GC-mediated control of adipose tissue browning. It is hoped that the plasticity of the adipose organ can be exploited in the next generation of therapeutic strategies to combat the increasing incidence of metabolic diseases, including obesity and diabetes.

Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

  • Berger CN
  • Cell Metab.
  • 2017 Nov 7

Literature context:


Abstract:

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.

Funding information:
  • NIDA NIH HHS - P01 DA031656(United States)

A Predictive Model for Selective Targeting of the Warburg Effect through GAPDH Inhibition with a Natural Product.

  • Liberti MV
  • Cell Metab.
  • 2017 Oct 3

Literature context:


Abstract:

Targeted cancer therapies that use genetics are successful, but principles for selectively targeting tumor metabolism that is also dependent on the environment remain unknown. We now show that differences in rate-controlling enzymes during the Warburg effect (WE), the most prominent hallmark of cancer cell metabolism, can be used to predict a response to targeting glucose metabolism. We establish a natural product, koningic acid (KA), to be a selective inhibitor of GAPDH, an enzyme we characterize to have differential control properties over metabolism during the WE. With machine learning and integrated pharmacogenomics and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE, leading to a therapeutic window in vivo. Thus, the basis of targeting the WE can be encoded by molecular principles that extend beyond the status of individual genes.

Funding information:
  • NCI NIH HHS - R00 CA168997()
  • NCI NIH HHS - R01 CA174643()
  • NCI NIH HHS - R01 CA193256()
  • NIDDK NIH HHS - R01 DK105550()
  • NIGMS NIH HHS - T32 GM007273()
  • NIGMS NIH HHS - T32 GM008500()

Fgf10-Hippo Epithelial-Mesenchymal Crosstalk Maintains and Recruits Lung Basal Stem Cells.

  • Volckaert T
  • Dev. Cell
  • 2017 Oct 9

Literature context:


Abstract:

The lung harbors its basal stem/progenitor cells (BSCs) in the protected environment of the cartilaginous airways. After major lung injuries, BSCs are activated and recruited to sites of injury. Here, we show that during homeostasis, BSCs in cartilaginous airways maintain their stem cell state by downregulating the Hippo pathway (resulting in increased nuclear Yap), which generates a localized Fgf10-expressing stromal niche; in contrast, differentiated epithelial cells in non-cartilaginous airways maintain quiescence by activating the Hippo pathway and inhibiting Fgf10 expression in airway smooth muscle cells (ASMCs). However, upon injury, surviving differentiated epithelial cells spread to maintain barrier function and recruit integrin-linked kinase to adhesion sites, which leads to Merlin degradation, downregulation of the Hippo pathway, nuclear Yap translocation, and expression and secretion of Wnt7b. Epithelial-derived Wnt7b, then in turn, induces Fgf10 expression in ASMCs, which extends the BSC niche to promote regeneration.

Funding information:
  • NHLBI NIH HHS - R01 HL092967()
  • NHLBI NIH HHS - R01 HL126732()
  • NHLBI NIH HHS - R01 HL132156()

P2Y6 regulates cytoskeleton reorganization and cell migration of C2C12 myoblasts via ROCK pathway.

  • Wang W
  • J. Cell. Biochem.
  • 2017 Aug 18

Literature context:


Abstract:

Migration of skeletal muscle precursor cells is required for limb muscle development and skeletal muscle repair. This study aimed to examine the role of P2Y6 receptor in C2C12 myoblasts migration. C2C12 myoblasts were treated with P2Y6 agonist UDP, P2Y6 antagonist MRS2578, Ca2+ channel blocker BTP2, or ROCK inhibitor GSK269962 or Y27632, and the migration ability of C2C12 cells was assessed by wound healing assay. The cellular Ca2+ content was analyzed with fluo-4 probe and the activation of ROCK (phosphorlyation of LIMK and cofilin) was assayed by western blot. The cytoskeleton was labeled with Actin-Tracker Green and Tubulin-Tracker-Red. Silencing P2Y6 expression in C2C12 myoblasts reduced intracellular Ca2+ content and cell motility. Whereas UDP increased cellular Ca2+ content, actin filaments, and cell migration, MRS2578 had the opposite effects. The effects of UDP were abrogated by BTP2 and GSK269962 (and Y27632). Disruption of P2Y6 signaling pathway caused C2C12 myoblasts to have an elongated morphology. These results demonstrated that P2Y6 signaled through Ca2+ influx and RhoA/ROCK to reorganize cytoskeleton and promote migration in myoblasts.

Polysialic Acid Regulates Sympathetic Outflow by Facilitating Information Transfer within the Nucleus of the Solitary Tract.

  • Bokiniec P
  • J. Neurosci.
  • 2017 Jul 5

Literature context:


Abstract:

Expression of the large extracellular glycan, polysialic acid (polySia), is restricted in the adult, to brain regions exhibiting high levels of plasticity or remodeling, including the hippocampus, prefrontal cortex, and the nucleus of the solitary tract (NTS). The NTS, located in the dorsal brainstem, receives constant viscerosensory afferent traffic as well as input from central regions controlling sympathetic nerve activity, respiration, gastrointestinal functions, hormonal release, and behavior. Our aims were to determine the ultrastructural location of polySia in the NTS and the functional effects of enzymatic removal of polySia, both in vitro and in vivo polySia immunoreactivity was found throughout the adult rat NTS. Electron microscopy demonstrated polySia at sites that influence neurotransmission: the extracellular space, fine astrocytic processes, and neuronal terminals. Removing polySia from the NTS had functional consequences. Whole-cell electrophysiological recordings revealed altered intrinsic membrane properties, enhancing voltage-gated K+ currents and increasing intracellular Ca2+ Viscerosensory afferent processing was also disrupted, dampening low-frequency excitatory input and potentiating high-frequency sustained currents at second-order neurons. Removal of polySia in the NTS of anesthetized rats increased sympathetic nerve activity, whereas functionally related enzymes that do not alter polySia expression had little effect. These data indicate that polySia is required for the normal transmission of information through the NTS and that changes in its expression alter sympathetic outflow. polySia is abundant in multiple but discrete brain regions, including sensory nuclei, in both the adult rat and human, where it may regulate neuronal function by mechanisms identified here.SIGNIFICANCE STATEMENT All cells are coated in glycans (sugars) existing predominantly as glycolipids, proteoglycans, or glycoproteins formed by the most complex form of posttranslational modification, glycosylation. How these glycans influence brain function is only now beginning to be elucidated. The adult nucleus of the solitary tract has abundant polysialic acid (polySia) and is a major site of integration, receiving viscerosensory information which controls critical homeostatic functions. Our data reveal that polySia is a determinant of neuronal behavior and excitatory transmission in the nucleus of the solitary tract, regulating sympathetic nerve activity. polySia is abundantly expressed at distinct brain sites in adult, including major sensory nuclei, suggesting that sensory transmission may also be influenced via mechanisms described here. These findings hint at the importance of elucidating how other glycans influence neural function.

Funding information:
  • NIEHS NIH HHS - R01 ES004738(United States)

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

  • Welsbie DS
  • Neuron
  • 2017 Jun 21

Literature context:


Abstract:

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Peripubertal Stress With Social Support Promotes Resilience in the Face of Aging.

  • Morrison KE
  • Endocrinology
  • 2017 Jun 5

Literature context:


Abstract:

The peripubertal period of development is a sensitive window, during which adverse experiences can increase the risk for presentation of cognitive and affective dysfunction throughout the lifespan, especially in women. However, such experiences in the context of a supportive social environment can actually ameliorate this risk, suggesting that resilience can be programmed in early life. Affective disorders and cognitive deficits commonly emerge during aging, with many women reporting increased difficulty with prefrontal cortex (PFC)-dependent executive functions. We have developed a mouse model to examine the interaction between peripubertal experience and age-related changes in cognition and stress regulation. Female mice were exposed to peripubertal chronic stress, during which they were either individually housed or housed with social interaction. One year after this stress experience, mice were examined in tasks to access their cognitive ability and flexibility in stress reactive measures. In a test of spatial memory acquisition and reversal learning where aged females normally display a decreased performance, the females that had experienced stress with social interaction a year earlier showed improved performance in reversal learning, a measure of cognitive flexibility. Because peripuberty is a time of major PFC maturation, we performed transcriptomic and biochemical analysis of the aged PFC, in which long-term changes in microRNA expression and in myelin proteins were found. These data suggest that stress in the context of social support experienced over the pubertal window can promote epigenetic reprogramming in the brain to increase the resilience to age-related cognitive decline in females.

Funding information:
  • NIMH NIH HHS - R21 MH098506(United States)

An Intrinsic Epigenetic Barrier for Functional Axon Regeneration.

  • Weng YL
  • Neuron
  • 2017 Apr 19

Literature context:


Abstract:

Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons' intrinsic axonal growth competence are not well understood. Here, we show that peripheral sciatic nerve lesion in adult mice leads to elevated levels of Tet3 and 5-hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, Tet3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a Tet3- and thymine DNA glycosylase-dependent fashion in DRG neurons. In addition, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult CNS is attenuated upon Tet1 knockdown. Together, our study suggests an epigenetic barrier that can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system.

Funding information:
  • NIGMS NIH HHS - T32 GM007814()

Intestinal Bile Acid Composition Modulates Prohormone Convertase 1/3 (PC1/3) Expression and Consequent GLP-1 Production in Male Mice.

  • Morimoto K
  • Endocrinology
  • 2016 Mar 27

Literature context:


Abstract:

Besides an established medication for hypercholesterolemia, bile acid binding resins (BABRs) present antidiabetic effects. Although the mechanisms underlying these effects are still enigmatic, glucagon-like peptide-1 (GLP-1) appears to be involved. In addition to a few reported mechanisms, we propose prohormone convertase 1/3 (PC1/3), an essential enzyme of GLP-1 production, as a potent molecule in the GLP-1 release induced by BABRs. In our study, the BABR colestimide leads to a bile acid-specific G protein-coupled receptor TGR5-dependent induction of PC1/3 gene expression. Here, we focused on the alteration of intestinal bile acid composition and consequent increase of total TGR5 agonistic activity to explain the TGR5 activation. Furthermore, we demonstrate that nuclear factor of activated T cells mediates the TGR5-triggered PC1/3 gene expression. Altogether, our data indicate that the TGR5-dependent intestinal PC1/3 gene expression supports the BABR-stimulated GLP-1 release. We also propose a combination of BABR and dipeptidyl peptidase-4 inhibitor in the context of GLP-1-based antidiabetic therapy.

Funding information:
  • NHLBI NIH HHS - 1K25HL68704(United States)
  • NIDDK NIH HHS - R01DK073911(United States)

Evidence for Vitamin D Receptor Expression and Direct Effects of 1α,25(OH)2D3 in Human Skeletal Muscle Precursor Cells.

  • Olsson K
  • Endocrinology
  • 2016 Jan 31

Literature context:


Abstract:

Presence of the vitamin D receptor and direct effects of vitamin D on the proliferation and differentiation of muscle precursor cells have been demonstrated in animal models. However, the effects and mechanisms of vitamin D actions in human skeletal muscle, and the presence of the vitamin D receptor in human adult skeletal muscle, remain to be established. Here, we investigated the role of vitamin D in human muscle cells at various stages of differentiation. We demonstrate that the components of the vitamin D-endocrine system are readily detected in human muscle precursor cells but are low to nondetectable in adult skeletal muscle and that human muscle cells lack the ability to convert the inactive vitamin D-metabolite 25-hydroxy-vitamin D3 to the active 1α,25-dihydroxy-vitamin D3 (1α,25(OH)2D3). In addition, we show that 1α,25(OH)2D3 inhibits myoblast proliferation and differentiation by altering the expression of cell cycle regulators and myogenic regulatory factors, with associated changes in forkhead box O3 and Notch signaling pathways. The present data add novel information regarding the direct effects of vitamin D in human skeletal muscle and provide functional and mechanistic insight to the regulation of myoblast cell fate decisions by 1α,25(OH)2D3.

Funding information:
  • NCATS NIH HHS - UL1 TR001082(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)

Gestational protein restriction impairs insulin-regulated glucose transport mechanisms in gastrocnemius muscles of adult male offspring.

  • Blesson CS
  • Endocrinology
  • 2014 Aug 19

Literature context:


Abstract:

Type II diabetes originates from various genetic and environmental factors. Recent studies showed that an adverse uterine environment such as that caused by a gestational low-protein (LP) diet can cause insulin resistance in adult offspring. The mechanism of insulin resistance induced by gestational protein restriction is not clearly understood. Our aim was to investigate the role of insulin signaling molecules in gastrocnemius muscles of gestational LP diet-exposed male offspring to understand their role in LP-induced insulin resistance. Pregnant Wistar rats were fed a control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery and a normal diet after weaning. Only male offspring were used in this study. Glucose and insulin responses were assessed after a glucose tolerance test. mRNA and protein levels of molecules involved in insulin signaling were assessed at 4 months in gastrocnemius muscles. Muscles were incubated ex vivo with insulin to evaluate insulin-induced phosphorylation of insulin receptor (IR), Insulin receptor substrate-1, Akt, and AS160. LP diet-fed rats gained less weight than controls during pregnancy. Male pups from LP diet-fed mothers were smaller but exhibited catch-up growth. Plasma glucose and insulin levels were elevated in LP offspring when subjected to a glucose tolerance test; however, fasting levels were comparable. LP offspring showed increased expression of IR and AS160 in gastrocnemius muscles. Ex vivo treatment of muscles with insulin showed increased phosphorylation of IR (Tyr972) in controls, but LP rats showed higher basal phosphorylation. Phosphorylation of Insulin receptor substrate-1 (Tyr608, Tyr895, Ser307, and Ser318) and AS160 (Thr642) were defective in LP offspring. Further, glucose transporter type 4 translocation in LP offspring was also impaired. A gestational LP diet leads to insulin resistance in adult offspring by a mechanism involving inefficient insulin-induced IR, Insulin receptor substrate-1, and AS160 phosphorylation and impaired glucose transporter type 4 translocation.

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

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)

Deficiency of ALADIN impairs redox homeostasis in human adrenal cells and inhibits steroidogenesis.

  • Prasad R
  • Endocrinology
  • 2013 Sep 26

Literature context:


Abstract:

Triple A syndrome is a rare, autosomal recessive cause of adrenal failure. Additional features include alacrima, achalasia of the esophageal cardia, and progressive neurodegenerative disease. The AAAS gene product is the nuclear pore complex protein alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN), of unknown function. Triple A syndrome patient dermal fibroblasts appear to be more sensitive to oxidative stress than wild-type fibroblasts. To provide an adrenal and neuronal-specific disease model, we established AAAS-gene knockdown in H295R human adrenocortical tumor cells and SH-SY5Y human neuroblastoma cells by lentiviral short hairpin RNA transduction. AAAS-knockdown significantly reduced cell viability in H295R cells. This effect was exacerbated by hydrogen peroxide treatment and improved by application of the antioxidant N-acetylcysteine. An imbalance in redox homeostasis after AAAS knockdown was further suggested in the H295R cells by a decrease in the ratio of reduced to oxidized glutathione. AAAS-knockdown SH-SY5Y cells were also hypersensitive to oxidative stress and responded to antioxidant treatment. A further impact on function was observed in the AAAS-knockdown H295R cells with reduced expression of key components of the steroidogenic pathway, including steroidogenic acute regulatory and P450c11β protein expression. Importantly a significant reduction in cortisol production was demonstrated with AAAS knockdown, which was partially reversed with N-acetylcysteine treatment. CONCLUSION: Our in vitro data in AAAS-knockdown adrenal and neuronal cells not only corroborates previous studies implicating oxidative stress in this disorder but also provides further insights into the pathogenic mechanisms in triple A syndrome.

Funding information:
  • NCRR NIH HHS - 2G12RR03060-26A1(United States)

A novel α-synuclein-GFP mouse model displays progressive motor impairment, olfactory dysfunction and accumulation of α-synuclein-GFP.

  • Hansen C
  • Neurobiol. Dis.
  • 2013 Aug 5

Literature context:


Abstract:

Compelling evidence suggests that accumulation and aggregation of alpha-synuclein (α-syn) contribute to the pathogenesis of Parkinson's disease (PD). Here, we describe a novel Bacterial Artificial Chromosome (BAC) transgenic model, in which we have expressed wild-type human α-syn fused to green fluorescent protein (GFP), under control of the mouse α-syn promoter. We observed a widespread and high expression of α-syn-GFP in multiple brain regions, including the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and the ventral tegmental area, the olfactory bulb as well as in neocortical neurons. With increasing age, transgenic mice exhibited reductions in amphetamine-induced locomotor activity in the open field, impaired rotarod performance and a reduced striatal dopamine release, as measured by amperometry. In addition, they progressively developed deficits in an odor discrimination test. Western blot analysis revealed that α-syn-GFP and phospho-α-syn levels increased in multiple brain regions, as the mice grew older. Further, we observed, by immunohistochemical staining for phospho-α-syn and in vivo by two-photon microscopy, the formation of α-syn aggregates as the mice aged. The latter illustrates that the model can be used to track α-syn aggregation in vivo. In summary, this novel BAC α-syn-GFP model mimics a unique set of aspects of PD progression combined with the possibility of tracking α-syn aggregation in neocortex of living mice. Therefore, this α-syn-GFP-mouse model can provide a powerful tool that will facilitate the study of α-syn biology and its involvement in PD pathogenesis.

Funding information:
  • NIDCD NIH HHS - 5R01DC000127(United States)
  • NIGMS NIH HHS - GM066826(United States)