1,721,008 research outputs found
Early intracellular events induced by in vivo leptin treatment in mouse skeletal muscle
No full textExperimental evidence suggests that leptin may exert direct effects on peripheral tissues. In this study we investigated some transductional molecules in skeletal muscle, after intraperitoneal leptin injection in wild-type and ob/ob mice. By immunoprecipitation and immunoblotting with anti-phosphotyrosine antibodies, we observed a modified pattern of phosphotyrosine proteins. We then identified an increase in JAK2, IRS1 and IRS2 tyrosine-phosphorylation and in their association with p85, a subunit of PI3K. The increase in PI3K activity in immunoprecipitated p85 did not reach statistical significance, however, both Akt and GSK3 resulted significantly hyper-phosphorylated. Bad, an Akt substrate involved in cell survival, appeared modified in its phosphorylation. ERK1, ERK2 and p38 MAP kinase phosphorylation significantly increased, even if the latter only in wild-type animals. Finally, by EMSA experiments, we documented that leptin increased the DNA binding capacity of Stat3 homodimers and AP-1. Thus, leptin appears to activate, within minutes, some insulin signalling molecules. Stat3 and AP-1 activation by gene expression remodelling could subsequently trigger more leptin-specific effects. Further, leptin might play a still underestimated role in cell survival
Caco-2/HT-29 cell co-culture mimicking the intestinal barrier is a tunable model for gut aging
No full textConsidering the physiological role played by the intestinal epithelial barrier (IEB), the research related to modifications due to the microbiota and intestinal cell alterations with aging is attracting more and more attention. The necessity to standardize the appropriate experimental models is still unmet and is accompanied by a critical need to develop an in vitro study model of the IEB reproducing the interactions between the absorptive and the secreting cells related to aging.
The present study aimed at characterizing the morphology and the physiology of the aged IEB through an in vitro model constituted by a co-culture of the two cell lines Caco-2 and HT-29 that we previously differentiated and characterized in absorptive and mucus-secreting cells respectively1,2. The co-culture was set up by plating a 70/30 ratio mixture of differentiated Caco2 cells from the 24th to 50th passage and HT-29 cells from the 8th to 35th passages for inducing “physiological” aging, in the absence of any exogenous stimulus. In the aged co-culture set up by plating Caco-cells which had reached at least 40th sub cultivation passage and HT-29 the 21st passage, we observed relevant morphofunctional impairments as i) a diminished epithelial electrical resistance (TEER); ii) an increased paracellular permeability; iii) a slight decrease in cell proliferation; and iv) a less homogeneous distribution of the membrane-associated claudin-1 immunostaining. Transmission electron microscopy (TEM) analysis revealed that the intracellular mucus and desmosomes were less represented in the aged co-culture, together with underdeveloped apical microvilli. These results suggest that this experimental setting can reproduce some of the main morphofunctional modifications of IEB reported in clinics in the leaky/aged gut. Future experiments could ascertain the use of the aged in vitro Caco-2 and HT-29 cell co-culture as a useful model for studying the molecular process and testing potential drug/nutraceutical treatments to ameliorate gut aging
Intracellular signal transduction pathways induced by leptin in C2C12 cells
No full textAs experimental evidence suggests that leptin may have direct effects on peripheral tissues, we investigated some of the transductional molecules induced by leptin in C2C12 cells. In immunoprecipitation experiments using anti-p85 antibodies (a regulatory subunit of phosphatidylinositol-3-kinase; PI3K), we observed a significant increase in PI3K activity. Immunoblot analyses showed that Akt, GSK3, ERK1, ERK2, and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation significantly increased after leptin treatment. Protein kinase C (PKC)-zeta was also activated by leptin, as documented by an immunocomplex kinase assay and immunoblotting experiments. The treatment of C2C12 cells with Wortmannin before leptin administration inhibited induction of the phosphorylation of ERKs (extracellular signal-regulated kinases) but not that of p38 MAPK, whereas pre-treatment with a PKC-zeta inhibitor partially decreased ERK phosphorylation. Taken together, our in vitro results further support the hypothesis that leptin acts acutely on skeletal muscle tissue through some of the components of insulin signalling, including PKC-zeta. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved
Signal transduction pathway of prolactin in rat liver
No full textWe previously reported that a single intraperitoneal injection of prolactin (PRL) in female adult rats rapidly and transiently activates mitogen-activated protein kinase (MAPK) in the liver. Here we analysed the PRL signalling pathway that accounts for MAPK activation. We found that total liver MAPK kinase-1 phosphorylating activity and Raf-1 activity significantly increase after PRL treatment, following a time course that accounts for the activation of MAPK. We also identified a significant increase in the phosphotyrosine content of the 52 kDa Shc protein, accompanied by an increase in Shc coimmunoprecipitated Grb2. which suggests the Ras involvement by PRL. We found that Janus kinase (JAK)2 tyrosine kinase, which appears constitutively associated with the PRL receptor expressed in the liver, is activated and associated with Shc proteins after in vivo PRL treatment. Taken together our data provide evidence that in vivo PRL activates the Shc-Ras-Raf-MAPK cascade in the liver by the involvement of JAK2 and suggests the possibility that the liver short form of PRL receptor plays a role in triggering this signalling pathway
Leptin rapidly activates PPARs in C2C12 muscle cells
No full textExperimental evidence suggests that leptin operates on the tissues, including skeletal muscle, also by modulating gene expression. Using electrophoretic mobility shift assays, we have shown that physiological doses of leptin promptly increase the binding of C2C12 cell nuclear extracts to peroxisome proliferator-activated receptor (PPAR) response elements in oligonucleotide probes and that all three PPAR isoforms participate in DNA-binding complexes. We pre-treated C2C12 cells with AACOCF(3), a specific inhibitor of cytosolic phospholipase A(2) (cPLA(2)), an enzyme that supplies ligands to PPARs, and found that it abrogates leptin-induced PPAR DNA-binding activity. Leptin treatment significantly increased cPLA(2) activity, evaluated as the release of [H-3]arachidonic acid from pre-labelled C2C12 cells, as well as phosphorylation. Further, using MEK1 inhibitor PD-98059 we showed that leptin activates cPLA(2) through ERK induction. These results support a direct effect of leptin on skeletal Muscle cells, and suggest that the hormone may modulate muscle transcription also by precocious activation of PPARs through ERK cPLA(2) pathway. (c) 2005 Elsevier Inc. All rights reserved
Rapid stimulation of mitogen-activated protein kinase of rat liver by prolactin
Full text linkIntraperitoneal prolactin administration to female rats caused a rapid and transient stimulation of hepatic mitogen-activated kinase (MAP kinase) activity measured in vitro as cytosolic phosphotransferase capacity towards two specific substrates. Myelin basic protein kinase activity of MAP kinase immunoprecipitates confirmed the specificity and magnified the prolactin effect. Immunoblot experiments with anti-(MAP kinase) and anti-phosphotyrosine antibodies showed changes in both electrophoretic mobility and phosphotyrosine content of 40 and 44 kDa isoenzymes suggesting that prolactin affects these isoforms. Concomitant with the increase in MAP kinase activity, prolactin induced tyrosine phosphorylation in a number of liver proteins, suggesting a rapid involvement of tyrosine kinases which might be correlated in some way with MAP kinase activation. Protein kinase C activity, which has been implicated in the regulation of MAP kinase and in mediating the prolactin effect, does not seem to participate in MAP kinase activation
A tunable in vitro Caco-2/HT-29 cell co-culture mimicking the intestinal barrier aging: a morphofunctional study
No full textThe intestinal epithelial barrier (IEB) of elderlies undergoes a myriad of changes due to the microbiota and intestinal cell alterations, which drive the feed-forward cycle of leaky gut, inflammaging, dampened cell renewal, and defective mucus production 1. Despite different in vivo and ex vivo studies 2 have shed some light on the age-associated impairment of IEB integrity and cytokine production, the involved molecular mechanisms are not completely understood. Thus, there is a critical need to develop an in vitro study model of the IEB reproducing the interactions between the absorptive and the secreting cells related to aging.
The present study aimed at characterizing the morphology and the physiology of the aged IEB through an in vitro model constituted by a co-culture of Caco2 and HT-29 cells, differentiated in absorptive and mucus-secreting phenotypes respectively 3, and sub-cultivated both in standard condition and for a longer time so that cell aging is gradually induced in a “physiological” way, avoiding the administration of exogenous stimuli. Preliminary results evidenced in the aged co-culture i) a diminished epithelial electrical resistance (TEER); ii) an increased paracellular permeability; iii) a slight decrease in cell proliferation; iv) a less homogeneous distribution of the membrane-associated claudin-1 immunostaining. Transmission electron microscopy (TEM) analysis revealed that the intracellular mucus and desmosomes were less represented in the aged co-culture, together with underdeveloped apical microvilli. Taken together, these preliminary results suggest the presence of impaired barrier integrity associated with a modulation of the morphological features mimicking the leaky/aged gut as reported in clinics. Future experiments could ascertain the use of the aged in vitro Caco-2 and HT-29 cell co-culture as a useful model for both studying the molecular process and testing potential drug/nutraceutical treatments to ameliorate gut aging
Rat liver eicosanoid synthesis during turpentine-induced inflammation
No full textSubcellular liver fractions from rats receiving a subcutaneous injection of turpentine, which causes a local inflammation, show an increased synthesis of Prostaglandin E2 and Prostaglandin F2 alpha which reaches a peak 90 minutes and 3 hours after treatment, respectively. Stimulation of phospholipase A2 activity of liver cell preparations seems to be responsible for the supply of arachidonic acid necessary to feed PG synthesis: this stimulation is accompanied by unchanged levels of diacylglycerol lipase, diacylglycerol kinase and protein kinase C activities and by an unchanged content of diacylglycerol in the liver tissue. This picture does not favour the hypothesis of an involvement of phospholipase C in the early stages after turpentine treatment. Determinations of GTP-ase activity in plasma membrane-rich liver preparations give ambiguous results, which do not allow any conclusion on the possible role of G-proteins in phospholipase A2 activation
The liver response to in vivo heat shock involves the activation of MAP kinases and RAF and the tyrosine phosphorylation of Shc proteins
No full textWe have investigated the mechanisms of signal transduction in the response of liver to heat shock in vivo. By immunoblot experiments we have shown that heat shock decreases the electrophoretic mobility of the 40 and 43 kDa mitogen activated protein kinases (MAPKs) and we have found a significant increase of MAPK activity measured as phosphotransferase capacity of both cytosolic extracts and MAPK immunoprecipitates. To elucidate the signalling pathway which accounts for MAPK activation, we focused our attention on its upstream factors, Raf and Ras. We have shown that, heat shock activates Raf-1 kinase and causes an increase in phosphotyrosine content of the 52 kDa Shc protein accompanied by an increment in the amount of coimmunoprecipitated Grb2. These findings provide the first evidence that the Ras-Raf-MAPK pathway is activated in liver during heat shock in vivo
State and activity of protein kinase C in postischemic reperfused liver
No full textWe have studied the activity and the phorbol-binding capacity of protein kinase C (PKC) in subcellular fractions, as well as the relative amount of the enzyme protein in rat livers reperfused after severe nonnecrogenic ischemia. Ischemia causes a significant decrease in PKC phosphotransferase activity in both membranes and cytosol which lasts long after the reestablishment of the blood flow. The phorbol-binding capacity of the membrane fraction shows the same behavior. The amount of PKC protein decreases during ischemia (-25%) but returns to normal after reperfusion more promptly than activity and binding capacity, suggesting that PKC resynthesized in postischemic livers is either functionally defective or incapacitated by unsuitable conditions of the environment. We have also measured the contents of some lipids that may influence PKC activity in the cell. During ischemia and reperfusion there is a significant increase in the content of 1,2-diacylglycerol (DAG), which is the physiological activator of PKC, but under the conditions occurring in the ischemic/postischemic livers DAG apparently cannot bind to the enzyme and fulfill its function. Total phospholipids, phosphatidylcholine, and phosphatidylethanolamine, which significantly decrease at 60 min of ischemia, return to normal levels 1 hr after reperfusion
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