Insulin stimulates the release of the glycosyl phosphatidylinositol-anchored membrane dipeptidase from 3T3-L1 adipocytes through the action of a phospholipase C.
Ontology highlight
ABSTRACT:
SUBMITTER: Movahedi S
PROVIDER: S-EPMC1218701 | biostudies-other | 1997 Sep
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.
| S-EPMC1137374 | biostudies-other
Project description:BackgroundHypoxia resulting from adipocyte expansion is considered the basis of the inflammatory milieu observed in Metabolic Syndrome. Nicotinic acid can act on adipocytes interfering on the inflammatory response. In this study, we investigated the role of HIF-1 α (hypoxia-inducible factor -1 alpha) in the inflammatory process induced by hypoxia. The effect of nicotinic acid on the PPARs (peroxisome proliferator-activated receptors) expression during the inflammatory response was assessed over its action under HIF-1 α in 3T3-L1 adipocytes submitted to hypoxia.Methods3T3-L1 adipocytes were pre-treated with nicotinic acid and incubated under hypoxic conditions. The level of adipokines and HIF-1 α were quantified using immunoassays. Adipokine expression was measured using real-time PCR, whereas PPARs and HIF-1 α expression were analyzed by western blot. The statistical significance of the differences between variables studied was determined by analysis of variance (ANOVA) complemented by Bonferroni's test.ResultsThe results demonstrated an increase in leptin and PAI-1 (plasminogen activator inhibitor-1) expression, while adiponectin production decreased under hypoxia. In parallel, induction with hypoxia enhanced HIF-1 α expression, despite causing reduced expression of PPAR α and PPAR γ. However, nicotinic acid reversed adipokine modulation under hypoxic conditions, leading to decreased HIF-1 α expression and increased PPARs expression.ConclusionsOur findings suggest that nicotinic acid blunt the inflammatory response resulting from hypoxia by the reduction of HIF-1 α expression and concomitant increase of PPARs α and γ expression in 3T3-L1 adipocytes.
| S-EPMC4862071 | biostudies-literature
Project description:Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.
| S-EPMC1149627 | biostudies-other
Project description:Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquat-induced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-dependent 2-deoxyglucose uptake. Under these conditions, oxidative stress did not affect insulin-dependent tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and -2, or binding of the phosphatidylinositol 3'-OH kinase (PI 3-kinase) p85 regulatory subunit or p110alpha catalytic subunit to each IRS. In contrast, we found that oxidative stress induced by paraquat inhibited activities of PI 3-kinase bound to IRSs and also inhibited phosphorylation of Akt, the downstream serine/threonine kinase that has been shown to play an essential role in insulin-dependent translocation of GLUT4 to the plasma membrane. Overexpression of active form Akt (myr-Akt) restored inhibition of insulin-dependent glucose uptake by paraquat, indicating that paraquat-induced oxidative stress inhibits insulin signals upstream of Akt. Paraquat treatment with and without insulin treatment decreased the activity of class Ia PI 3-kinases p110alpha and p110beta that are mainly expressed in 3T3-L1 adipocytes. However, paraquat treatment did not repress the activity of the PI 3-kinase p110alpha mutated at Cys(90) in the p85 binding region. These results indicate that the PI 3-kinase p110 is a possible primary target of paraquat-induced oxidative stress to reduce the PI 3-kinase activity and impaired glucose uptake in 3T3-L1 adipocytes.
| S-EPMC2898352 | biostudies-literature
Project description:Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.
| S-EPMC9272592 | biostudies-literature
Project description:The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.
| S-EPMC1220670 | biostudies-literature
Project description:A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20-30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5-7 min at 37 degrees C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.
| S-EPMC30573 | biostudies-literature
Project description:Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin-Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.
| S-EPMC1218411 | biostudies-other
Project description:Perfluorobutanesulfonic acid (PFBS) is used as the replacement of perfluorooctanesulfonic acid (PFOS) since 2000 because of the concern on PFOS' persistence in the environment and the bioaccumulation in animals. Accumulating evidence has shown the correlation between the exposure to perfluorinated compounds and enhanced adipogenesis. There is no report, however, of the effect of PFBS on adipogenesis. Therefore, the present work aimed to investigate the role of PFBS in adipogenesis using 3T3-L1 adipocytes. PFBS treatment for 6 days extensively promoted the differentiation of 3T3-L1 preadipocytes to adipocytes, resulting in significantly increased triglyceride levels. In particular, the treatments of PFBS at the early adipogenic differentiation period (day 0-2) were positively correlated with increased the triglyceride accumulation on day 6. PFBS treatments significantly increased the protein and mRNA levels of the master transcription factors in adipocyte differentiation; CCAAT/enhancer-binding protein ? (C/EBP?) and peroxisome proliferator-activated receptor gamma (PPAR?), along with acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), the key proteins in lipogenesis. PFBS significantly activated the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) after 4-h treatment, and PFBS' effect on triglyceride was abolished by U0126, a specific MAPK/ERK kinase (MEK) inhibitor. In conclusion, PFBS increased the adipogenesis of 3T3-L1 adipocytes, in part, via MEK/ERK-dependent pathway.
| S-EPMC6169790 | biostudies-literature
Project description:Exposure to endocrine disrupting chemicals is an established risk factor for obesity. The most commonly used pesticide active ingredients have never been tested in an adipogenesis assay. We tested for the first time the potential of glyphosate, 2, 4-dichlorophenoxyacetic acid, dicamba, mesotrione, isoxaflutole, and quizalofop-p-ethyl (QpE) to induce lipid accumulation in murine 3T3-L1 adipocytes. Only QpE caused a dose-dependent statistically significant triglyceride accumulation from a concentration of 5 up to 100 µM. The QpE commercial formulation Targa Super was 100 times more cytotoxic than QpE alone. Neither the estrogen receptor antagonist ICI 182, 780 nor the glucocorticoid receptor antagonist RU486 was able to block the QpE-induced lipid accumulation. RNAseq analysis of 3T3-L1 adipocytes exposed to QpE suggests that this compound exerts its lipid accumulation effects via a peroxisome proliferator-activated receptor gamma (PPARγ)-mediated pathway, a nuclear receptor whose modulation influences lipid metabolism. QpE was further shown to be active in a PPARγ reporter gene assay at 100 µM, reaching 4% of the maximal response produced by rosiglitazone, which acts as a positive control. This indicates that lipid accumulation induced by QpE is only in part caused by PPARγ activation. The lipid accumulation capability of QpE we observe suggest that this pesticide, whose use is likely to increase in coming years may have a hitherto unsuspected obesogenic property.
| S-EPMC6657571 | biostudies-literature