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1.
The mechanism responsible for the stimulation of secretion of lipoprotein lipase by heparin in cultured cells was studied with avian adipocytes in culture. Immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radiolabeled enzyme, whereas total lipoprotein lipase was quantitated by radioimmunoassay. Rates of synthesis of lipoprotein lipase were not different for control or heparin treatments as judged by incorporation of L-[35S]methionine counts into lipoprotein lipase during a 20-min pulse. This observation was corroborated in pulse-chase experiments where the calculation of total lipoprotein lipase synthesis, based on the rate of change in enzyme-specific activity during the chase, showed no difference between control (8.13 +/- 3.1) and heparin treatments (9.1 +/- 5.3 ng/h/60-mm dish). Secretion rates of enzyme were calculated from measurements of the radioactivity of the secreted enzyme and the cellular enzyme-specific activity. Degradation rates were calculated by difference between synthesis and secretion rates of enzyme. In control cells 76% of the synthesized enzyme was degraded. Addition of heparin to the culture medium reduced the degradation rate to 21% of the synthetic rate. The presence of heparin in cell media resulted in a decrease in apparent intracellular retention half-time for secreted enzyme from 160 +/- 44 min to 25 +/- 1 min. The above data demonstrate that the increase in lipoprotein lipase protein secretion, observed upon addition of heparin to cultured adipocytes, is due to a decreased degradation rate with no change in synthetic rate. Finally, newly synthesized lipoprotein lipase in cultured adipocytes is secreted constitutively and there is no evidence that it is stored in an intracellular pool. 相似文献
2.
Poly(A)+ mRNA isolated from chicken adipose tissue directed cell-free translation in a rabbit reticulocyte lysate system. Immunoadsorption with polyclonal antibodies against lipoprotein lipase detected a protein of 56 +/- 2 kDa. Immunodetection of this protein was prevented by inclusion of purified lipoprotein lipase in the assay mixture. Identification of the 56 kDa protein as lipoprotein lipase was confirmed by immunoadsorption to the monoclonal antibody CAL 1-11. Inclusion of dog pancreatic microsomal membranes in the translation system resulted in isolation of an additional protein of 62 kDa. Treatment of the 62 kDa protein with endo-beta-N-acetylglycosaminidase H or endo-beta-N-acetylglucosaminidase F decreased the observed molecular mass to that of the primary translation product, indicating that the increase in molecular mass resulted from the addition of N-linked oligosaccharides. Starving and refeeding chickens prior to poly(A)+ mRNA isolation resulted in a 3-fold increase in the amount of immunodetectable lipoprotein lipase synthesized. 相似文献
3.
The reciprocal regulation of lipoprotein lipase activity and hormone-sensitive lipase activity in rat adipocytes 总被引:9,自引:0,他引:9
R L Patten 《The Journal of biological chemistry》1970,245(21):5577-5584
4.
Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes 总被引:2,自引:0,他引:2
The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate. 相似文献
5.
H H Klein G R Freidenberg S Matthaei J M Olefsky 《The Journal of biological chemistry》1987,262(22):10557-10564
We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization. 相似文献
6.
Insulin rapidly increases glycerol-3-phosphate-acyltransferase activity in rat adipocytes. 总被引:1,自引:0,他引:1
Glycerol-3-phosphate acyltransferase (G3PAT) was activated by insulin in intact rat adipocytes within 1 min: this activation persisted for 10 min, and was due to a decrease in the Km of the enzyme. The addition of insulin to control adipocyte membranes also increased G3PAT activity, and this effect was mimicked by phosphatidylinositol-specific phospholipase C. Cytosol fractions from insulin-treated adipocytes stimulated G3PAT activity of control membranes, suggesting that a soluble mediator is released during insulin action, possibly through activation of a PI-specific PLC. 相似文献
7.
Ballart X Siches M Peinado-Onsurbe J López-Tejero D Llobera M Ramírez I Robert MQ 《Biochimie》2003,85(10):971-982
White adipose tissue (WAT) lipoprotein lipase (LPL) activity channels diet fat towards storage in adipocytes. Adrenaline (ADR) is accepted to reduce WAT or adipocyte LPL activity (LPLa), but available data are not clear-cut regarding long exposure to ADR in vitro or in vivo. We studied the effects of long exposures to ADR or beta-adrenergic agonist on LPL: in isolated rat adipocytes (3 h) and in rats (>1 day). Isoproterenol (ISO) (1 microM) did not alter LPLmRNA nor LPLa in adipocytes, but increased LPLa in medium more than twofold (3.58 +/- 0.35 vs. 1.32 +/- 0.35 mU/10(6) adipocytes, P < 0.001). Effect was time (not present at 1 h, clear at 2 h) and concentration dependent (high sensitivity from 10 to 100 nM, max at 1 microM). Adenylate cyclase activator or cyclic AMP (cAMP) analogue produced a similar increase. Thus in adipocytes ISO produced an increase in LPLa release and/or a decrease in extracellular LPLa degradation. ADR or ISO treated rats had a two to fourfold decrease in WAT LPLa vs. unchanged LPLmRNA. This decrease was 10-fold in WAT heparin-releasable LPLa (5.7 +/- 0.6 vs. 57.3 +/- 10.2 mU/g, P < 0.001), which represents peri/extracellular LPLa. Plasma LPLa was increased 11-fold by ADR (3.30 +/- 0.58 vs. 0.32 +/- 0.08 mU/ml, P < 0.001) whereas only threefold by ISO (P > 0.01). We suggest that in vivo ADR increased release of active LPL to plasma from endothelial cells of LPL-rich tissue(s)-WAT was probably one of these tissues releasing LPL since it lost 90% of its peri/extracellular LPLa-and/or decreased degradation of plasma active LPL. Since liver LPLa was not increased, plasma active LPL might be kept away from hepatic degradation by binding to stabilising entities in plasma (fatty acids (FA), lipoproteins or soluble heparan sulphates (HS)). In conclusion, we believe this is the first report stating that: (a) ISO increases LPLa in isolated adipocyte medium, and (b) ADR administration to rats decreases WAT extracellular active LPL and increases preheparin plasma active LPL. 相似文献
8.
PPAR-gamma,TNF-alpha messenger RNA levels and lipase activity in the pregnant and lactating rat 总被引:2,自引:0,他引:2
Dramatic alternations in maternal metabolism occur during gestation and lactation, especially glucose and fat metabolism. For example, in rats, the amount of body fat mass increases during gestation, then decreases just prior to delivery, and remains low after parturition. To investigate the factors involved in such changes in maternal fat mass, messenger ribonucleic acid (mRNA) levels of adipocytokines, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and tumor necrosis factor-alpha (TNF-alpha), were examined in the intraabdominal adipose tissue of non-pregnant rats, pregnant rats and postpartum rats. We also examined the issue of whether apoptosis, which could be promoted by PPAR-gamma and TNF-alpha, is involved in any of the changes in maternal fat mass The activity of lipoprotein lipase (LPL) and hormone sensitive lipase (HSL) in adipose tissue was also measured. PPAR-gamma and TNF-alpha mRNA levels remained constant during the gestational and postpartum periods. Apoptosis was not detected at any time as evidenced by DNA laddering and in situ staining. LPL activity was significantly increased at day 5 and remained elevated until day 14 of gestation. HSL activity was significantly increased at day 10 of gestation and then decreased after delivery, at day 10 of lactation. In conclusion, during the gestational and postpartum period of rats, changes in maternal fat mass did not directly correlate with the levels of expression of PPAR-gamma and TNF-alpha mRNA. Apoptosis also does not appear to influence on fat mass change. The changes in LPL and HSL activities during gestation suggest that these enzymes might be regulators of maternal adipose tissue level. 相似文献
9.
The role of glucose and glycosylation in the regulation of lipoprotein lipase synthesis and secretion in rat adipocytes 总被引:4,自引:0,他引:4
Several studies have suggested that insulin and glucose increase adipose tissue lipoprotein lipase (LPL). To study the mechanism of the glucose-induced stimulation of LPL, the effects of glucose and glycosylation were examined in primary rat adipocyte cultures. In cells cultured in the presence of 1 mg/ml glucose, a 55-kDa LPL protein was synthesized and secreted into the medium, whereas cells cultured in glucose-free medium synthesized a 49-kDa form of LPL which was not secreted. The treatment of the mature 55-kDa form of LPL with peptide:N-glycosidase-F resulted in the formation of a 49-kDa form of LPL. When cells were cultured in the presence of tunicamycin, a 49-kDa form of LPL was synthesized by the cells but was not secreted. In addition, LPL activity was reduced by 90% when glycosylation was blocked by either tunicamycin or glucose deprivation. LPL synthetic rate was examined in cells cultured in a spectrum of glucose concentrations. LPL synthetic rate increased directly with medium glucose concentration and was decreased 80% in the absence of glucose compared to the synthetic rate in the presence of 1 mg/ml glucose. In addition, LPL synthetic rate in the presence of insulin was approximately 200% of the synthetic rate in untreated control cells at all glucose concentrations and even in the absence of glucose. In spite of the effect of glucose on LPL synthetic rate, glucose had no effect on the level of LPL mRNA. In contrast, the mRNA for the 78-kDa glucose-regulated protein (GRP78) was increased in adipocytes cultured in glucose-free medium. In summary, glucose was essential for glycosylation of LPL, and glycosylation was essential for LPL catalytic activity and secretion. In addition, glucose stimulated LPL synthetic rate and potentiated the stimulatory effects of insulin, but had no specific effect on LPL mRNA. Whereas insulin stimulates LPL by increasing the level of LPL mRNA, glucose stimulates LPL translation and post-translational processing. 相似文献
10.
Takashi Yokota Mikio Nagashima Mohammad Ghazizadeh Oichi Kawanami 《Life sciences》2009,84(15-16):523-529
AimsFucoidan, a sulfated polysaccharide extracted from brown seaweed (F. vesiculosus) is recognized as an effective anticoagulant but its anti-lipidemic potency has not been well defined. We investigated the effect of fucoidan on lipoprotein lipase (LPL) secretion by human adipocytes.Main methodsLPL mRNA and protein expressions were measured using semi-quantitative RT-PCR, ELISA and immunohistochemistry in cultured adipocytes with or without fucoidan treatment. LPL enzyme activity was determined by a fluorometric assay.Key findingsIn cultured adipocytes, fucoidan induced LPL secretion in a dose- and time-dependent manner. An initial increase in LPL was maintained at a significant level but much slower than that in heparin-treated cells. Fucoidan also dose-dependently induced a cofactor of LPL, the apolipoprotein C-II (ApoC-II) secretion. In fucoidan-treated cells, LPL mRNA was time-dependently increased and LPL protein expression was also inceased. Treatment with both heparin and fucoidan showed no further increase in media LPL activity compared to heparin alone. In the conditioned medium from fucoidan-treated cells followed for 4 h, LPL activity decayed exponentially with half-life of about 180 min. In addition, the extracellular LPL mass in cycloheximide (a protein synthesis inhibitor) and fucoidan-treated cells did not change markedly, but LPL shifted significantly from active to inactive form.SignificanceThese results suggest that fucoidan acts like heparin by releasing LPL in addition to increasing the intracellular transport and decreasing the degradation of LPL in the medium. Furthermore, LPL and ApoC-II secretion induced by fucoidan may be involved in regulating plasma triglyceride lowering clearance. 相似文献
11.
Guinea-pig adipocytes released lipoprotein lipase activity to the medium without depletion of cell-associated lipoprotein lipase activity. Heparin caused immediate release of 20-25% of the lipase activity to the medium, and also enhanced the continued release. After addition of cycloheximide, cell-associated lipoprotein lipase activity decreased rapidly. Release of lipase activity to the medium continued unabated for about 30 min, but there was little release thereafter. The release accounted for only about 25% of the initial lipoprotein lipase activity in the absence and about 50% in the presence of heparin. In pulse-chase experiments with [35S]methionine, labeled lipoprotein lipase appeared in the medium within 40 min, and most of the release occurred during the first h of chase. In a 4-h chase the total (cells + medium) amount of labeled lipase decreased to 34%. Thus, degradation was a main fate of the lipase. Heparin markedly increased the amount of labeled lipase that was released to the medium and decreased the amount that was degraded. Heparin did not change the time-course for the release, and the amount of labeled lipase degraded was proportional to the amount not released to the medium, indicating that the effect of heparin was primarily on release, not on degradation as such. This study demonstrates that adipocytes synthesize lipoprotein lipase in excess of what is being released, and that the excess is rapidly degraded. 相似文献
12.
13.
Prolonged treatment of adipocytes with certain inhibitors of lipolysis, including N(6)-phenylisopropyl adenosine (PIA) and prostaglandin E(1) (PGE(1)) leads to down-regulation of G(i). Prolonged treatment with PIA increases the rate of lipolysis, and we have reported that tumor necrosis factor-alpha (TNF alpha) stimulates lipolysis by down-regulating G(i). To determine the relationship between G(i) concentration and lipolysis, we have investigated the effect of two other acute inhibitors of lipolysis; PGE(1), which down-regulates G(i), and nicotinic acid (NA), which does not down-regulate G(i). Rat adipocytes were incubated with PIA (300 nM), PGE(1) (3 microM) or nicotinic acid (1 mM) for 24 h. The rate of lipolysis (glycerol release) was increased approximately 2 to 3-fold in PIA-treated cells, and in PGE(1)-treated cells. Conversely, the rate of lipolysis was not altered by the prolonged nicotinic acid treatment. These findings support the hypothesis that the rate of lipolysis in adipocytes is determined, at least partly, by the cellular concentration of G(i) proteins. 相似文献
14.
Recombinant human tumor necrosis factor does not inhibit lipoprotein lipase in primary cultures of isolated human adipocytes 总被引:2,自引:0,他引:2
P A Kern 《Journal of lipid research》1988,29(7):909-914
Previous studies have demonstrated that cachectin/tumor necrosis factor (TNF) inhibits lipoprotein lipase (LPL) activity in cultures of 3T3-L1 cells. To determine whether TNF also inhibits LPL in human adipocytes, primary cultures of isolated human adipocytes were exposed to a spectrum of concentrations of recombinant human TNF. TNF concentrations up to 1000 pM had no effect on either LPL activity or LPL immunoreactive mass in the human adipocytes. Specific binding of 125I-labeled TNF was demonstrated in human adipocytes, and a TNF concentration of 100 pM competed for approximately 50% of the 125I-labeled TNF binding sites. In contrast, the same TNF in the same concentrations progressively inhibited LPL activity and immunoreactive mass in 3T3-L1 cells. Thus, human adipocytes respond to TNF in a different manner than 3T3-L1 cells. TNF may not cause the cachexia of cancer or chronic infection by directly inhibiting LPL in adipose tissue. 相似文献
15.
16.
17.
Vanadate increased lipoprotein lipase (LPL) activity in the isolated fat pads in a time- and dose-dependent manner. The increasing effect of vanadate was inhibited by amiloride, similar to that of insulin, and it also was not additive to that of insulin. Although the increasing effects of vanadate and insulin were preserved in K(+)-free medium, appreciable decreases in both effects were observed by replacement of Na+ with choline ion or omission of Ca2+ in the medium. Vanadate showed the full effect in the presence of cycloheximide at concentrations that inhibited protein synthesis of the fat pads, suggesting that the action of vanadate is not due to the increase in protein synthesis. Tetrakis (acetoxymethyl) ester of quin 2 at 50 microM concentration never inhibited the action of vanadate though it showed a little inhibition at a concentration of 300 microM. No inhibition of the action of vanadate was observed with ruthenium red. These results suggest that vanadate increases the LPL activity via a process less sensitive to the intracellular Ca2+ concentration. Adrenaline, dibutyryl cyclic AMP, and 3-isobutyl-1-methylxanthine all inhibited the action of vanadate, suggesting that the action is inhibited with increase in the intracellular concentration of cyclic AMP. Monensin and carbonyl cyanide m-chlorophenylhydrazone inhibited the action of vanadate. In contrast, the action of insulin was never inhibited by monensin. Tunicamycin and 2-deoxyglucose, at rather high concentrations, inhibited both actions. These findings suggest that vanadate increases the LPL activity through mechanisms of action involving amiloride- and monensin-sensitive pathways dependent on energy. 相似文献
18.
Summary Heparin (5 U/ml) induced the release of LPL into the incubation medium of cardiac myocytes isolated from adult rat hearts. The secretion of LPL occurred in two phases: a rapid release (5–10 min of incubation with heparin) that was independent of protein synthesis followed by a slower rate of release that was inhibited by cycloheximide. The rapid release of LPL induced by heparin likely occurs from sites that are at or near the cell surface. LPL secretion could also be stimulated by heparan sulfate and dermatan sulfate, but not by hyaluronic acid, chondroitin sulfate or keratan sulfate. Heparin-releasable LPL activity measured in short-term incubations represented a large fraction (40–50%) of the initial LPL activity associated with myocytes, but the fall in cellular LPL activity following heparin was less than the amount of LPL activity secreted into the incubation medium. This discrepancy was not due to latency of LPL in the pre-heparin cell homogenates, but in part could be due to a three-fold greater affinity of the heparin-released enzyme for substrate as compared to LPL in post-heparin myocyte homogenates.Abbreviations LPL
lipoprotein lipase 相似文献
19.
Insulin processing and signal transduction in rat adipocytes 总被引:1,自引:0,他引:1
A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction. 相似文献
20.
The lipoprotein lipase (clearing-factor lipase) activity of cells isolated from rat cardiac muscle. 总被引:2,自引:2,他引:0
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The total lipoprotein lipase activity recovered in suspension of cells prepared from adult rat hearts was unaffected by the nutritional state of the animals used. The enzyme activity present in the cell suspensions was almost exclusively associated with the cardiac muscle cells present as the major cell type. 相似文献