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1.
The incorporation of long-chain fatty acids into phospholipids has been detected in adipocyte ghosts that were incubated with [1-14 C] stearic, [1-14 C] linoleic or [1-14 C] arachidonic acid. Adrenaline and adenosine activated this incorporation within 15 s of exposure of the ghosts to the hormones and the response was dose dependent. Maximum incorporation of labelled linoleic acid occurred at 10(-5) M adrenaline and 10(-7) M adenosine. The alpha-agonist phenylephrine and the beta-agonist isoproterenol were also shown to stimulate the incorporation of fatty acid in a dose dependent manner. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were each labelled preferentially with linoleic or arachidonic acid. p-Bromophenacylbromide, quinacrine and centrophenoxine inhibited the adrenaline-stimulated incorporation of fatty acids into ghost membrane phospholipids, and p-bromophenacylbromide also reduced the activation of adenylate cyclase by adrenaline. NaF, an activator of adenylate cyclase, like adrenaline, stimulated the incorporation of linoleic acid into ghost membrane phospholipids.  相似文献   

2.
Progesterone induced a transient increase in the incorporation of [3H]methyl groups into phospholipids of Xenopus oocytes followed by a rise in 45Ca2+ uptake. Phospholipid methylation reached a maximum as early as 15 s after progesterone treatment and returned to basal level within 2 min. Steroids inactive in promoting oocyte maturation were less effective in affecting phospholipid methylation. Methyltransferase inhibitors, 3-deaza-SIBA, SIBA, and Sinefungin, inhibited progesterone-activated stimulation of phospholipid methylation, calcium uptake and meiotic maturation. Phospholipid methylation is the earliest detectable biochemical event occurring in oocytes after exposure to progesterone followed by calcium influx and leading to germinal vesicle dissolution.  相似文献   

3.
Intact platelets actively incorporate 3H-methionine and successively methylate phosphatidylethanolamine to 3H-phosphatidyl- N-monomethylethanolamine (PME), 3H-phosphatidyl-N, N-dimethylethanolamine (PDE) and 3H-phosphatidylcholine (PC) in platelet membranes. Phospholipid methylation is dependent on time, temperature, pH and methionine concentration. Thrombin, epinephrine and adenine potently inhibit phospholipid methylation and to a lesser degree enhance degradation of 3H-methylated phospholipids. Unlike 3H-methylated phospholipids in red cells and synaptosomes, 3H-PME, 3H-PDE and 3H-PC are symmetrically distributed on both sides of platelet membranes. Furthermore, in contrast to leukocytes, methylation derived PC is not distinguishable from CDP-choline derived PC as a substrate for arachidonic acid release since platelets labelled with 3H-methionine, 3H-choline and 14C-arachidonic acid all showed similar degradation of labelled PC when stimulated with thrombin. 3H-S-adenosyl-L-methionine (SAM) is not actively incorporated by intact platelets; however, lysed platelet membrane fragments were able to utilize 3H-SAM as a methyl donor. Addition of exogenous phospholipids enhanced product formation.  相似文献   

4.
5.
In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na+>Li+>K+>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na+>Li+>K+>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E1 on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E1, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.  相似文献   

6.
Summary The effects of theophylline on insulin receptors and insulin action in isolated rat adipocytes were studied. Theophylline reduced insulin binding by a decrease of receptor affinity. As concentration-response curves revealed, the effect was paralleled by a reduction of the cellular ATP content. Basal as well as insulin-stimulated glucose transport (2-deoxyglucose and 3-O-methylglucose uptake) were inhibited by much smaller theophylline concentrations (0.15–0.6 mM ) than those necessary to reduce insulin binding and to lower ATP levels (1–4.8 mM), or to stimulate lipolysis (0.3-2.4 mM). Insulin fully antagonized the effect of theophylline on lipolysis but failed to reverse the inhibition of glucose transport completely. The results suggest that (a) theophylline impairs insulin action at a post-receptor level and, at higher concentrations, by a decrease of receptor binding, (b) the reduction of insulin receptor affinity probably reflects ATP depletion of the adipocyte, and (c) the xanthine inhibits glucose transport independently from its effects on lipolysis.  相似文献   

7.
8.
Phospholipid methylation by hepatic microsomes was measured following glucagon or epinephrine administration either to intact rats or to the isolated perfused liver. Both hormones stimulated the methylation measured as the incorporation of S-adenosyl-L-[methyl-3H]methionine into phospholipids. The labeled products were identified by thin layer chromatography and most of the counts were found to be incorporated into phosphatidylcholine. The stimulatory effects of the hormones were evident already 5 minutes following hormone administration both in in vivo and in in vitro. The observed stimulation of the methylation process by glucagon and epinephrine might be related to the previously reported stimulatory effect of these hormones on the microsomal Ca2+-ATPase, and indicate that methylation process(es) might mediate some of the effects of these hormones.  相似文献   

9.
Regulation of adipocyte differentiation and insulin action with rapamycin   总被引:6,自引:0,他引:6  
Here, we demonstrated that inhibition of mTOR with rapamycin has negative effects on adipocyte differentiation and insulin signaling. Rapamycin significantly reduced expression of most adipocyte marker genes including PPARgamma, adipsin, aP2, ADD1/SREBP1c, and FAS, and decreased intracellular lipid accumulation in 3T3-L1 and 3T3-F442A cells, suggesting that rapamycin would affect both lipogenesis and adipogenesis. Contrary to the previous report that suppressive effect of rapamycin on adipogenesis is limited to the clonal expansion, we revealed that its inhibitory effect persisted throughout the process of adipocyte differentiation. Thus, it is likely that constitutive activation of mTOR might be required for the execution of adipogenic programming. In differentiated 3T3-L1 adipocytes, chronic treatment of rapamycin blunted the phosphorylation of AKT and GSK, which is stimulated by insulin, and reduced insulin-dependent glucose uptake activity. Taken together, these results suggest that rapamycin not only prevents adipocyte differentiation by decrease of adipogenesis and lipogenesis but also downregulates insulin action in adipocytes, implying that mTOR would play important roles in adipogenesis and insulin action.  相似文献   

10.
In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E1, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn2+ and Mg2+, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn2+ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E1 (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn2+, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E1?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn2+, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn2+ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg2+, although about 100-fold higher concentrations of Mg2+ than of Mn2+ were required. The data indicate that Mn2+at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn2+ on the functions of the guanine nucleotide site(s) in the system.  相似文献   

11.
Pref-1 is a highly glycosylated Delta-like transmembrane protein containing six epidermal growth factor-like repeats in the extracellular domain. Pref-1 is abundantly expressed in preadipocytes, but expression is down-regulated during adipocyte differentiation. Forced expression of Pref-1 in 3T3-L1 cells was reported to inhibit adipocyte differentiation. Here we show that efficient and regulated processing of Pref-1 occurs in 3T3-L1 preadipocytes releasing most of the extracellular domain as a 50-kDa heterogeneous protein, previously isolated and characterized as FA1. Unexpectedly, we found that forced expression of the soluble form, FA1, or full-length Pref-1 did not inhibit adipocyte differentiation of 3T3-L1 cells when differentiation was induced by standard treatment with methylisobutylxanthine, dexamethasone, and high concentrations of insulin. However, forced expression of either form of Pref-1/FA1 in 3T3-L1 or 3T3-F442A cells inhibited adipocyte differentiation when insulin or insulin-like growth factor-1 (IGF-1) was omitted from the differentiation mixture. We demonstrate that the level of the mature form of the IGF-1 receptor is reduced and that IGF-1-dependent activation of p42/p44 mitogen-activated protein kinases (MAPKs) is compromised in preadipocytes with forced expression of Pref-1. This is accompanied by suppression of clonal expansion and terminal differentiation. Accordingly, supplementation with insulin or IGF-1 rescued p42/p44 MAPK activation, clonal expansion, and adipocyte differentiation in a dose-dependent manner.  相似文献   

12.
Factor H of the human complement system exhibits an unusual circular dichroism spectrum. The CD spectrum of Factor H exhibits a positive extreme at 230 nm and a negative extreme at 190 nm. No apparent alpha-helical or beta-sheet conformations were present in the native protein structure. However, when the disulfide bridges are reduced, followed either by reoxidation or alkylation, the structure of Factor H is modified so that it now exhibits conventional protein secondary structure as determined from its CD spectra in the far ultraviolet region. Factor H also fails to mediate its regulatory function of inhibiting the alternative pathway convertase once the disulfides have been ruptured and conformational rearrangement has occurred. CD studies indicate that minor conformational changes take place when Factor H and C3b associate in free solution.  相似文献   

13.
We have assessed the influence of nondisplaceable (internalized) insulin and insulin degradation during binding reactions at 37 degrees C on the numbers and affinities of insulin binding sites on isolated rat adipocytes. Corrections for nondisplaceable insulin caused a 33% reduction in the number of the high affinity sites (p less than 0.01) and a 24% reduction (p less than 0.01) in the number of the low affinity sites which was associated with a 20% increase (p less than 0.01) in affinity when a two-site model was applied. With a one-site model, the number of insulin receptors decreased by approximately 33% (p less than 0.01), but the affinity did not change. These results indicate that the internalization and degradation of insulin that occurs during the binding reaction can significantly affect the estimation of insulin binding kinetics. Potential variations in internalization and degradation of insulin by cells obtained under various physiological or pathologic conditions should, therefore, be taken into consideration in the interpretation of insulin binding data.  相似文献   

14.
Calcium signals and phospholipid methylation in eukaryotic cells.   总被引:1,自引:3,他引:1  
Rat basophil leukaemic (2H3) cells, mast cells and mouse thymocytes respond to stimulation by specific ligands with an increase in the free cytosolic Ca2+ concentration. The time courses of these Ca signals and the biological responses have been compared with changes in phospholipid metabolism. Increased phosphoinositide metabolism coincides with the Ca signals and the responses in each cell system, whereas any increase in phospholipid methylation during the response is less than one molecule per receptor and at least 5-50-fold less than the increases reported previously. Furthermore, no significant changes were detected in the concentration of S-adenosylmethionine, the methyl-group donor in the synthesis of methylated phospholipids. The hypothesis that phospholipid methylation is obligatory for receptor-mediated Ca signals is not supported by these data and requires critical re-evaluation.  相似文献   

15.
16.
Insulin receptor molecules in rat adipocyte plasma membranes were shown to be monovalent with respect to their capacity to bind insulin. The 1:1 stoichiometry for insulin binding was determined by a "double-probe labeling" procedure, wherein 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess saturating concentration of an unlabeled biotinylated insulin derivative (probe 2). If the receptor were competent to bind more than one insulin molecule, any receptor molecule that was cross-linked to probe 1 also should have been cross-linked to probe 2 in the double probe labeling procedure. The monovalent character of the insulin receptor was indicated by the failure of the probe 1-linked receptor to be cross-linked to probe 2. This was indicated by the failure of succinylavidin to increase the molecular weight of the probe 1-linked receptor. Control experiments indicated that succinylavidin increased the molecular weight of receptor that had been cross-linked to probe 2. The 1:1 stoichiometry for insulin binding demonstrated here indicates that if insulin receptors contain more than one insulin binding subunit, the binding of insulin to its receptor must be a highly negatively cooperative process.  相似文献   

17.
18.
DNA methylation is an epigenetic mark on the mammalian genome. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences distributed throughout the genome. To determine the epigenetic changes during adipocyte differentiation, we investigated the sequential changes in DNA methylation status of 3T3-L1 cells at the growing, confluent, postconfluent and mature adipocyte cell stages. Treatment of 3T3-L1 cells with 5-aza-2′-deoxycytidine inhibited differentiation in a stage-dependent manner, supporting the idea that formation of accurate DNA methylation profile, consisting of methylated and unmethylated T-DMRs, may be involved in differentiation. Analysis by methylation-sensitive quantitative real-time PCR of the 65 known T-DMRs which contain NotI sites detected 8 methylations that changed during differentiation, and the changes in the patterns of these methylations were diverse, confirming that the differentiation process involves epigenetic alteration at the T-DMRs. Intriguingly, the dynamics of the methylation change vary depending on the T-DMRs and differentiation stages. Restriction landmark genomic scanning detected 32 novel T-DMRs, demonstrating that differentiation of 3T3-L1 cells involves genome-wide epigenetic changes by temporal methylation/demethylation, in addition to maintenance of a static methylated/demethylated state, and both depend on differentiation stage.  相似文献   

19.
It is usually accepted that GPEtn is only an intermediate in glycerophospholipid degradation. However, some years ago J. P. Infante published a review-hypothesis concerning the possible involvement of GPEtn and GPCho in the biosynthesis of acyl-specific glycerophospholipids in mammalian tissues. This paper reports the results obtained by incubating brain cortex slices in the presence of labeled GPEtn. GPEtn enters the cells and the radioactivity of the precursor is found in both PE and PC. When these results were compared with those obtained by incubating brain slices in the presence of3H-Etn, a significantly higher amount of radioactivity was found in PC, mainly after short incubations in the presence of low concentrations of the precursors. The slopes of the methylated forms of PE further underline the above differences found by comparing the results obtained with the two precursors. The results presented in this paper suggest a possible role of GPEtn in phospholipid biosynthesis and methylation pathway of PE.  相似文献   

20.
Insulin receptor cycling and insulin action in the rat adipocyte   总被引:4,自引:0,他引:4  
The possibility that the insulin receptor of adipocytes undergoes cycling was examined by a method involving pronase digestion at 12 degrees C, followed by insulin binding studies to determine receptor location and quantity. In the absence of insulin treatment, the amount of internal receptors (i.e. protected from pronase) was 10% of total receptor content. Following a 30-min insulin treatment (0.1 microM) at 37 degrees C, the internal receptor content increased 2-fold (206 +/- 12% of control, 100%). This effect was rapid, and maximum internalization was approached by 5 min of insulin treatment. Warming pronase-digested cells to 37 degrees C allowed the internal receptors to move to the cell surface. This movement was rapid also, and expansion of the internal pool by insulin pretreatment provided a 2.4-fold increase in the reinsertion of cell-surface receptors (238 +/- 28% of nontreated cells, 100%). Insulin-pretreated and nontreated cells had approximately 13 and 6%, respectively, of their original cell-surface receptor content, i.e. their content before pronase digestion. These receptors appeared intact after the cycling process, as judged by affinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor and its binding subunit. The ability of the recycled receptor to respond to insulin was examined by studies of glucose incorporation into lipids and the inhibition of isoproterenol-stimulated lipolysis. Cells pretreated with insulin and allowed to recycle (e.g. 13% of normal receptor content) were 2-3-fold more responsive and 7-fold more sensitive to subsequent insulin stimulation than nontreated cells (e.g. 6% of normal receptor content), indicating that the recycled receptors are biologically active and coupled to cellular effector systems.  相似文献   

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