scholarly journals The lipoprotein lipase of white adipose tissue. Changes in the adipocyte cell-surface content of enzyme in response to extracellular effectors in vitro

1986 ◽  
Vol 238 (1) ◽  
pp. 239-246 ◽  
Author(s):  
A A Al-Jafari ◽  
A Cryer
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
Lipoprotein Lipase ◽  
Protein Transport ◽  
Independent Effect ◽  
Adipocyte Cell ◽  
Tissue Changes ◽  
Insulin Dependent ◽  
Hormonal Action

An indirect labelled-second-antibody cellular immunoassay for adipocyte surface lipoprotein lipase was used to assess the changes that occurred during the incubation of cells in the presence and absence of effectors. In the absence of any specific effectors, the amount of immunodetectable lipoprotein lipase present at the surface of adipocytes remained constant throughout the 4 h incubation period at 37 degrees C. Under such conditions total cellular enzyme activity also remained constant, with no activity appearing in the medium. In the presence of heparin, cell-surface immunodetectable lipoprotein lipase increased by up to 20%, whereas in the presence of cycloheximide they decreased by up to 60%. Thus the obvious turnover of enzyme from this cell-surface site was found to be relatively rapid and dependent for its replenishment, at least in part, on protein synthesis. In the presence of insulin alone, a substantial increase in cell-surface lipoprotein lipase protein occurred, only part of which was dependent on protein synthesis. The total cellular activity of lipoprotein lipase was unaffected by the presence of insulin. The insulin-dependent increase in cell-surface enzyme was potentiated somewhat in the presence of dexamethasone, which was not shown to exert any independent effect. Glucagon, adrenaline and theophylline all produced a significant decline in the cell-surface immunodetectable lipoprotein lipase, which in the case examined (adrenaline) was partially additive with regard to the independent effect of cycloheximide. Cell-surface immunodetectable lipoprotein lipase amounts were decreased significantly when cells were incubated in the presence of either colchicine or tunicamycin. The concerted way in which cell-surface lipoprotein lipase altered during the incubations of adipocytes in the presence of effectors suggested that the translocation of enzyme to and from this cellular site was dependent on hormonal action and the integrity of intracellular protein-transport mechanisms.

scholarly journals Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.

1995 ◽  
Vol 129 (4) ◽  
pp. 999-1006 ◽  
Author(s):  
K V Kandror ◽  
J M Stephens ◽  
P F Pilch
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Buoyant Density ◽  
Fat Cells ◽  
Structural Protein ◽  
Coordinate Regulation ◽  
Rat Adipocytes ◽  
Transport Vesicles ◽  
Adipocyte Cell ◽  
Insulin Dependent

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin-sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.

Lipoprotein lipase binding to adipocytes: evidence for the presence of a heparin-sensitive binding protein

1993 ◽  
Vol 265 (6) ◽  
pp. E880-E888 ◽  
Author(s):  
A. Sasaki ◽  
P. Sivaram ◽  
I. J. Goldberg
Keyword(s):  
Cell Surface ◽  
Lipoprotein Lipase ◽  
Binding Protein ◽  
Surface Protein ◽  
Surface Binding ◽  
Cell Membrane Proteins ◽  
Adipocyte Cell ◽  
Capillary Endothelial Cells ◽  
Fold Excess ◽  
Ligand Blot

Lipoprotein lipase (LPL) is synthesized by adipocytes, associated with the cell surface, and released from the cells when they are treated with heparin. Release of LPL from the adipocyte is required for LPL to migrate to its physiological site of action on the luminal surface of capillary endothelial cells. To better understand this process, we studied the interaction of LPL with adipocyte cell membrane proteins. With the use of a ligand blot method, LPL specifically bound to a heparin-releasable, 116-kDa protein on mouse-derived brown fat adipose cell (BFC-1 beta) and rat adipocyte membranes. A 116-kDa cell surface protein was metabolically labeled with [35S]methionine and bound to LPL-Sepharose. This suggested that the LPL-binding protein was synthesized by the cells. When BFC-1 beta were treated with heparin to eliminate heparin-sensitive cell surface binding sites, LPL binding to the cells decreased and release of newly synthesized LPL activity increased. 125I-labeled LPL binding to control cells was reduced (> 70%) by a 50-fold excess of unlabeled LPL. The residual LPL binding to heparin-treated cells was, however, not decreased by the addition of unlabeled LPL. These data imply that specific adipocyte surface LPL binding involves heparin-sensitive sites. We hypothesize that the heparin-releasable, 116-kDa LPL-binding protein mediates specific LPL binding to adipocytes and that LPL activity within adipose tissue is regulated, in part, by the interaction of LPL with this binding protein.

scholarly journals The Fission Yeast Ran Gtpase Is Required for Microtubule Integrity

2000 ◽  
Vol 151 (5) ◽  
pp. 1101-1112 ◽  
Author(s):  
Ursula Fleig ◽  
Sandra S. Salus ◽  
Inga Karig ◽  
Shelley Sazer
Keyword(s):  
Fission Yeast ◽  
Protein Transport ◽  
Nucleocytoplasmic Transport ◽  
Independent Effect ◽  
Loss Of Function ◽  
Microtubule Cytoskeleton ◽  
Ran Gtpase ◽  
Yeast Genes

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.

scholarly journals Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

1994 ◽  
Vol 5 (7) ◽  
pp. 819-828 ◽  
Author(s):  
Y Wang ◽  
G M Fuller
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
De Novo ◽  
Cell Types ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Synthesis Inhibitors ◽  
Different Cell Types ◽  
Kinetics Of

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

scholarly journals Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells

1980 ◽  
Vol 188 (3) ◽  
pp. 941-944 ◽  
Author(s):  
P N Dalrymple ◽  
L L Houston
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
Indirect Immunofluorescence ◽  
Cultured Cells ◽  
Sodium Dithionite ◽  
Eukaryotic Cells ◽  
Protein Synthesis Inhibition ◽  
Inhibiting Protein ◽  
A Chain

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.

scholarly journals Primary Human Derived Blood Outgrowth Endothelial Cells: An Appropriate In Vitro Model to Study Shiga Toxin Mediated Damage of Endothelial Cells

Toxins ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 483
Author(s):  
Wouter J. C. Feitz ◽  
Nicole C. A. J. van de Kar ◽  
Ian Cheong ◽  
Thea J. A. M. van der Velden ◽  
Carolina G. Ortiz-Sandoval ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Protein Synthesis ◽  
Cell Surface ◽  
Shiga Toxin ◽  
Dependent Manner ◽  
Surface Binding ◽  
Necrosis Factor Alpha ◽  
Static Conditions

Hemolytic uremic syndrome (HUS) is a rare disease primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Endothelial damage is the hallmark of the pathogenesis of HUS with an infection with the Shiga toxin (Stx) producing Escherichia coli (STEC-HUS) as the main underlying cause in childhood. In this study, blood outgrowth endothelial cells (BOECs) were isolated from healthy donors serving as controls and patients recovered from STEC-HUS. We hypothesized that Stx is more cytotoxic for STEC-HUS BOECs compared to healthy donor control BOECs explained via a higher amount of Stx bound to the cell surface. Binding of Shiga toxin-2a (Stx2a) was investigated and the effect on cytotoxicity, protein synthesis, wound healing, and cell proliferation was studied in static conditions. Results show that BOECs are highly susceptible for Stx2a. Stx2a is able to bind to the cell surface of BOECs with cytotoxicity in a dose-dependent manner as a result. Pre-treatment with tumor necrosis factor alpha (TNF-α) results in enhanced Stx binding with 20–30% increased lactate dehydrogenase (LDH) release. Endothelial wound healing is delayed in a Stx2a-rich environment; however, this is not caused by an effect on the proliferation rate of BOECs. No significant differences were found between control BOECs and BOECs from recovered STEC-HUS patients in terms of Stx2a binding and inhibition of protein synthesis.

AMP-activated protein kinase activator A-769662 is an inhibitor of the Na+-K+-ATPase

2009 ◽  
Vol 297 (6) ◽  
pp. C1554-C1566 ◽  
Author(s):  
Boubacar Benziane ◽  
Marie Björnholm ◽  
Louise Lantier ◽  
Benoit Viollet ◽  
Juleen R. Zierath ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Cell Surface ◽  
Atpase Activity ◽  
Sodium Pump ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Independent Effect ◽  
Amp Activated Protein Kinase

Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AMPK). AMPK restores energy balance by activating processes that produce energy while inhibiting those that consume energy. The role of AMPK in the regulation of active ion transport is unclear. Our aim was to determine the effect of the AMPK activator A-769662 on Na+-K+-ATPase function in skeletal muscle cells. Short-term incubation of differentiated rat L6 myotubes with 100 μM A-769662 increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in parallel with decreased Na+-K+-ATPase α1-subunit abundance at the plasma membrane and ouabain-sensitive 86Rb+ uptake. Notably, the effect of A-769662 on Na+-K+-ATPase was similar in muscle cells that do not express AMPK α1- and α2-catalytic subunits. A-769662 directly inhibits the α1-isoform of the Na+-K+-ATPase, purified from rat and human kidney cells in vitro with IC50 57 μM and 220 μM, respectively. Inhibition of the Na+-K+-ATPase by 100 μM ouabain decreases sodium pump activity and cell surface abundance, similar to the effect of A-769662, without affecting AMPK and ACC phosphorylation. In conclusion, the AMPK activator A-769662 inhibits Na+-K+-ATPase activity and decreases the sodium pump cell surface abundance in L6 skeletal muscle cells. The effect of A-769662 on sodium pump is due to direct inhibition of the Na+-K+-ATPase activity, rather than AMPK activation. This AMPK-independent effect on Na+-K+-ATPase calls into question the use of A-769662 as a specific AMPK activator for metabolic studies.

scholarly journals Effects of insulin, glucocorticoids and adrenaline on the activity of rat adipose-tissue lipoprotein lipase

1980 ◽  
Vol 188 (1) ◽  
pp. 185-192 ◽  
Author(s):  
P Ashby ◽  
D S Robinson
Keyword(s):  
Protein Synthesis ◽  
Adipose Tissue ◽  
Lipoprotein Lipase ◽  
Lipase Activity ◽  
Lipoprotein Lipase Activity ◽  
Fat Bodies ◽  
Rat Adipose Tissue ◽  
Adipose Tissue Lipoprotein Lipase

The lipoprotein lipase activity of epididymal fat-bodies from starved rats was measured during incubations at 37 degrees C in vitro. Protein synthesis independent activation of the enzyme, previously observed during incubations at 25 decrease C, also occurs at 37 degrees C. Protein-synthesis-dependent increases in the activity of the enzyme occur in the presence of insulin and are markedly potentiated by glucocorticoids. The effects on the activity of the enzyme of insulin alone, or in the presence of glucocorticoids, are correlated with its effects on total protein synthesis in the tissue. Adrenaline antagonizes the increase in activity of the enzyme brought about by insulin and abolishes the potentiation of insulin action by glucocorticoids. These changes may be due, at least in part, to its stimulation of inactivation of the enzyme in the tissue. It is suggested that changes in adipose-tissue lipoprotein lipase activity that occur with changes in nutritional status in vivo result from the combined effects of changes in plasma insulin and glucocorticoid concentrations.

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