Leptin biosynthetic pathway in white adipocytes

2006 ◽  
Vol 84 (2) ◽  
pp. 207-214 ◽  
Author(s):  
Philippe G Cammisotto ◽  
Ludwik J Bukowiecki ◽  
Yves Deshaies ◽  
Moise Bendayan
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
De Novo ◽  
Brefeldin A ◽  
Mrna Levels ◽  
De Novo Synthesis ◽  
Cellular Content ◽  
Constitutive Secretion

The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum–Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.Key words: leptin, vesicles, constitutive secretion, de novo synthesis, transcription.

Ultrastructural changes in the rough endoplasmic reticulum and its contents following Brefeldin A treatment of human chondrocytes in vitro

1991 ◽  
Vol 49 ◽  
pp. 256-257
Author(s):  
H. E. Gruber
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Brefeldin A ◽  
Ultrastructural Level ◽  
Human Chondrocytes ◽  
Ultrastructural Changes ◽  
Ultrastructural Studies

The rough endoplasmic reticulum (rER) is now recognized as a major organelle responsible for ensuring that only structurally correct and properly folded proteins are allowed to enter the cellular secretory pathway. We are especially interested in the behavior of the chondrocyte rER since ultrastructural studies of many skeletal dysplasias have revealed that electron dense material accumulates or is not degraded within the rER of chondrocytes from patients. Remodelling of the rER in chick chondrocytes has also been evaluated at the ultrastructural level and the rER found to play a role in procollagen export from the cell. We have utilized normal human chondrocytes grown in culture to investigate the role of brefeldin A, an antiviral antibiotic, which has been shown to primarily block protein transport from the ER to the Golgi complex.

scholarly journals Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the Secretory Pathway, Respectively

2001 ◽  
Vol 75 (22) ◽  
pp. 10787-10799 ◽  
Author(s):  
Jason M. Mackenzie ◽  
Edwin G. Westaway
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Envelope Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Immunogold Labeling ◽  
Virus Particles ◽  
Infected Cells ◽  
Assembly Site

ABSTRACT The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.

scholarly journals Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

2006 ◽  
Vol 81 (2) ◽  
pp. 558-567 ◽  
Author(s):  
George A. Belov ◽  
Nihal Altan-Bonnet ◽  
Gennadiy Kovtunovych ◽  
Catherine L. Jackson ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Brefeldin A ◽  
Rna Replication ◽  
Bound State ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Virus Growth ◽  
Guanine Nucleotide Exchange ◽  
Membrane Reorganization

ABSTRACT Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

Bioactive Alkaloids from the Tropical Marine Sponge Axinella carteri

1995 ◽  
Vol 50 (9-10) ◽  
pp. 669-674 ◽  
Author(s):  
A. Supriyono ◽  
B. Schwarz ◽  
V. Wray ◽  
L. Witte ◽  
W. E. G. Müller ◽  
...  
Keyword(s):  
Marine Sponge ◽  
Insecticidal Activity ◽  
De Novo ◽  
The Philippines ◽  
De Novo Synthesis ◽  
Mouse Lymphoma Cells ◽  
Guanidine Alkaloids ◽  
Neonate Larvae ◽  
Mouse Lymphoma

Abstract Analysis of the tropical marine sponge Axinella carteri afforded six unusual alkaloids, including the new brominated guanidine derivative 3-bromo-hymenialdisine. The structure elucidation of the new alkaloid is described. The alkaloid patterns of sponges collected in Indonesia or in the Philippines were shown to be qualitatively identical suggesting de novo synthesis by the sponge or by endosymbiontic microorganisms rather than uptake by filterfeeding. All alkaloids were screened for insecticidal activity as well as for cytotoxicity. The guanidine alkaloids hymenialdisine and debromohymenialdisine exhibited insecticidal activity towards neonate larvae of the polyphagous pest insect Spodoptera littoralis (LD50s of 88 and 125 ppm, respectively), when incorporated into artificial diet and offered to the larvae in a chronic feeding bioassay. The remaining alkaloids, including the new compound, were inactive in this bioassay. Cytotoxicity was studied in vitro using L5178y mouse lymphoma cells. Debromohymenialdisine was again the most active compound (ED50 1.8 μg/ml) followed by hymenialdisine and 3-bromohymenialdisine, which were essentially equitoxic and exhibited ED50s of 3.9 μg/ml in both cases. The remaining alkaloids were inactive against this cell line

Immunology: Evidence for the in-vitro de-novo synthesis of immunoglobulin and a previously undescribed 17 kDa protein (TEP-2) by the mucosa of the Fallopian tube

1993 ◽  
Vol 8 (8) ◽  
pp. 1199-1202 ◽  
Author(s):  
S.D. Maguiness ◽  
K. Shrimanker ◽  
O. Djahanbakhch ◽  
B. Teisner ◽  
J.G. Grudzinskas
Keyword(s):  
Fallopian Tube ◽  
De Novo ◽  
De Novo Synthesis

scholarly journals ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro

1999 ◽  
Vol 147 (7) ◽  
pp. 1457-1472 ◽  
Author(s):  
Chung-Chih Lin ◽  
Harold D. Love ◽  
Jennifer N. Gushue ◽  
John J.M. Bergeron ◽  
Joachim Ostermann
Keyword(s):  
Secretory Pathway ◽  
Mutant Cell ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Secretory Proteins ◽  
Intermediate Step ◽  
Golgi Stack ◽  
Transport Vesicles ◽  
Direct Fusion

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

Intrapulmonary catabolism of surfactant-saturated phosphatidylcholine in rabbits

1990 ◽  
Vol 69 (5) ◽  
pp. 1856-1862 ◽  
Author(s):  
E. D. Rider ◽  
M. Ikegami ◽  
A. H. Jobe
Keyword(s):  
Alveolar Macrophages ◽  
Secretory Pathway ◽  
De Novo ◽  
Turnover Time ◽  
Lung Homogenate ◽  
De Novo Synthesis ◽  
Phospholipase A1 ◽  
Pool Sizes

Intrapulmonary surfactant catabolism was investigated by use of a phospholipase A1- and A2-resistant analogue of dipalmitoylphosphatidylcholine (DPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPC ether). [14C]DPC ether, made into liposomes with [3H]DPC and associated with 32P-labeled rabbit surfactant, was given intratracheally to 1-kg rabbits, which were killed at preset times to 48 h. Recoveries of radiolabel as saturated phosphatidylcholine (Sat PC) isolated from alveolar wash (AW), postlavage lung homogenate (LH), and alveolar macrophages were measured. All groups had similar AW and LH Sat PC pool sizes, indicating no perturbation of endogenous Sat PC pools. Despite a nearly fivefold accumulation of [14C]DPC ether in the lung by 48 h (P less than 0.01), the three probes had similar alveolar clearance curves. Furthermore, the Sat PC reutilization efficiency (41.6%) and turnover time (5.9 h) calculated for DPC ether were not different from values for the DPC and rabbit surfactant. Of the DPC ether (0.7%) and DPC (9%) labels recovered as PC in organs outside the lung, greater than 85% was unsaturated, indicating de novo synthesis using precursors from degraded PC. DPC ether was a useful probe of intrapulmonary DPC catabolism, and after alveolar uptake there was no direct reentry of intact DPC from the catabolic compartment(s) into the secretory pathway.

scholarly journals Locations and contributions of the phosphotransferases EPT1 and CEPT1 to the biosynthesis of ethanolamine phospholipids

2020 ◽  
Vol 61 (8) ◽  
pp. 1221-1231 ◽  
Author(s):  
Yasuhiro Horibata ◽  
Hiromi Ando ◽  
Hiroyuki Sugimoto
Keyword(s):  
Fatty Acids ◽  
De Novo ◽  
Brefeldin A ◽  
Hek293 Cells ◽  
Intracellular Location ◽  
De Novo Biosynthesis ◽  
Substrate Preferences ◽  
Ethanolamine Phosphate ◽  
Immunohistochemical Analyses

The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/EPT1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for PE production and 1-alkyl-2-acyl-glycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine. Here, we investigated the intracellular location and contribution to ethanolamine phospholipid (EP) biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the ER. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamine disclosed that EPT1 is more important for the de novo biosynthesis of 1-alkenyl-2-acyl-glycerophosphoethanolamine than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did not significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16–20:4 > DAG 18:0–20:4 > DAG 16:0–18:1 = AAG 16–18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0–18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.

scholarly journals Release of interleukin-1 and interleukin-6 from human monocytes by antithymocyte globulin: requirement for de novo synthesis

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2531-2538 ◽  
Author(s):  
P Rameshwar ◽  
P Gascon
Keyword(s):  
Antithymocyte Globulin ◽  
De Novo ◽  
Interleukin 1 ◽  
Immunosuppressive Agent ◽  
Biologically Active ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hematopoietic Growth Factors

Abstract Antithymocyte globulin (ATG) is an effective treatment in patients with severe aplastic anemia (SAA). Its mechanism of action remains unclear, although it has been assumed to be immunosuppressive. However, ATG has also been shown by several laboratories to be immunostimulatory. Recently, interleukin-1 (IL-1) production has been found to be decreased in lipopolysaccharide-stimulated peripheral blood monocytes obtained from SAA patients. We have investigated the ability of ATG to function as an immunostimulatory agent via the production of IL-1 and IL-6 by normal human monocytes in vitro. Supernatants from ATG- stimulated monocytes were assayed for biologically active and immunoreactive IL-1 and IL-6. We have found that ATG, via its F(ab')2 fragment is a powerful inducer of IL-1 and IL-6 production. Furthermore, ATG induction of both cytokines from normal monocytes required de novo synthesis, as determined by 35S-methionine incorporation. Because these two cytokines synergize with other cytokines at both the stem cell and progenitor levels, these stimulatory properties of ATG may be relevant to the treatment of SAA. This would favor the hypothesis of a bimodal mechanism for ATG as an inducer of hematopoietic growth factors and as an immunosuppressive agent.

scholarly journals Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor

1983 ◽  
Vol 3 (8) ◽  
pp. 1362-1370 ◽  
Author(s):  
H. Bussey ◽  
D. Saville ◽  
D. Greene ◽  
D. J. Tipper ◽  
K. A. Bostian
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Killer Toxin ◽  
Membrane System ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Endoproteolytic Cleavage ◽  
Toxin Secretion

Killer toxin secretion was blocked at the restrictive temperature inSaccharomyces cerevisiae secmutants with conditional defects in theS. cerevisiaesecretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in allsecmutants at the restrictive temperature. Insec18the protoxin was stable after a chase; but insec7andsec1the protoxin was unstable, and insec111K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and insec7andsec1cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined twokexmutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable inkex1but unstable inkex2. Protoxin stability inkex1 kex2double mutants indicated the orderkex1→kex2in the protoxin processing pathway. TPCK did not block protoxin instability inkex2mutants. This suggested that theKEX1- andKEX2-dependent steps preceded thesec7Golgi block. We attempted to localize the protoxin inS. cerevisiaecells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

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