31 research outputs found
Activation of Protein Kinase A and Exchange Protein Directly Activated by cAMP Promotes Adipocyte Differentiation of Human Mesenchymal Stem Cells
Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I2 (PGI2) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells
Coexistence of honeybees with distinct mitochondrial haplotypes and hybridised nuclear genomes on the Comoros Islands
Sublethal effect assessment of a low-power and dual-frequency anti-cyanobacterial ultrasound device on the common carp (Cyprinus carpio): a field study
Adipocyte differentiation of hMADS cells requires the activation of both Epac- and PKA-dependent signaling pathways.
<p>Two-day post-confluent hMADS cells were induced to differentiate with 1 µM Dex, 0.86 µM insulin (DI), in the presence of 200 µM of the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP (007) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034114#pone.0034114-Christensen1" target="_blank">[37]</a>, 100 µM of PKA selective cAMP analog 6-MB-cAMP (MB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034114#pone.0034114-Christensen1" target="_blank">[37]</a>, either separately or in combination (007+MB) from day 0 to day 3. Rosi (0.5 µM) was present from day 3–9. Undifferentiated hMADS cells were taken as control. (A) The panel shows cells on day 14 stained with Oil-Red-O. The photographs and micrographs shown are representative of 3 independent experiments. (B) RNA was isolated on day 14, and expression of <i>PPARG2</i>, <i>CEBPA</i>, <i>FABP4</i> and <i>LPL</i> was determined by RT-qPCR. Date presented were normalized to the value of DI treated cells. Significant differences are indicated by asterisks, *p<0.05, **p<0.01, ***p<0.001, n = 9. (C) Whole cell extracts were prepared and analyzed for FABP4 protein level by Western blotting. (D and E) Two-day post-confluent hMADS cells were treated for 15 min with 200 µM 8-pCPT-2′-O-Me-cAMP (007) or 100 µM 6-MB-cAMP (MB) or an equal volume of water (vehicle) in medium with Dex and Insulin. (D) GTP bound RAP1 was measured by a RAP1 activation pull-down assay as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034114#s4" target="_blank">Materials and Methods</a>”. (E) PKA activity in cell lysates was determined as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034114#s4" target="_blank">Materials and Methods</a>”. Significant differences relative to DI treated cells are indicated by asterisks, *p<0.05, **p<0.01, ***p<0.001, n = 4.</p
Expression of <i>Epac</i> and <i>Rap</i> mRNAs in hMADS cells.
<p>Cells were differentiated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034114#s4" target="_blank">Materials and Methods</a>”. RNA was isolated on the indicated days. The levels of <i>RAPGEF3 (Epac1)</i>, <i>RAPGEF4 (Epac2)</i> mRNAs (A) and <i>RAP1A</i>, <i>RAP1B</i>, <i>RAP2A</i>, <i>RAP2B</i>, <i>RAP2C</i> mRNAs (B) were determined by RT-qPCR. Data presented in (A) were normalized to <i>RAPGEF3</i> on day 0; in (B) data were normalized to <i>RAP1B</i> on day0. Significant differences are indicated by asterisks, *p<0.05, **p<0.01, ***p<0.001, n = 8.</p
Dominant negative Epac1 attenuates adipocyte differentiation of hMADS cells.
<p>hMADS cells were infected with retroviruses expressing a dominant-negative form of Epac1 (dnEpac1) or an empty vector and grow to confluence. (A) GTP-bound Rap1 was measured by a Rap1 activation pull-down assay. (B) Cells were induced to differentiate with Dex, insulin, IBMX and Rosi as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034114#s4" target="_blank">Materials and Methods</a>”. On day 14, cells were stained with Oil-Red-O and photographed. The photographs and micrographs shown are representative of 4 independent experiments. (C) <i>PPARG2</i>, <i>CEBPA</i>, <i>FABP4</i> and <i>LPL</i> mRNA levels were determined by RT-qPCR. Expression was normalized to cells transduced with empty vector. Significant differences are indicated by asterisks, **p<0.01, ***p<0.001, n = 6.</p
A cAMP elevating agent is required for adipocyte differentiation of hMADS cells.
<p>(A) Two-day post-confluent hMADS cells were induced to differentiate with 1 µM Dex and 0.86 µM insulin (DI) in the presence or absence of 0.5 mM IBMX, 100 µM 8-pCPT-cAMP or an equal volume of water (vehicle) from day 0 to day 3. Thereafter, 0.5 µM rosi was added until day 9. On day 14, cells were fixed, and cytoplasmic triglycerides were stained with Oil-Red-O. Undifferentiated hMADS cells were used as control. The photographs and micrographs shown are representative of 3 independent experiments. (B) RNA was isolated on day 14, and expression of <i>PPARG2</i>, <i>CEBPA</i>, <i>FABP4</i> and <i>LPL</i> was determined by RT-qPCR. Data presented were normalized to the value of DI treated cells. Significant differences relative to DI treated cells are indicated by asterisks, *p<0.05, **p<0.01, ***p<0.001, n = 6.</p
Carbaprostacyclin rescues adipocyte differentiation in the absence of other cAMP elevating agents.
<p>Two-day post-confluent hMADS cells were maintained in induction media with 0.86 µM insulin in the presence or absence of 1 µM Dex, 0.5 mM IBMX or 0.2 or 1 µM carbaprostacyclin (cPGI) as indicated from day 0 to day 3. Thereafter, 0.5 µM rosiglitazone was added until day 9. GPDH activities were determined on day 14.</p
Effects of PPAR agonists and long-chain fatty acids on adipocyte differentiation of hMADS cells.
<p>Two-day post-confluent hMADS cells were maintained in serum-free DMEM/Ham's F12 medium in the presence of 1 µM Dex, 0.5 mM IBMX, and 0.86 µM insulin. From day 3, the medium consisted of DMEM/Ham's F12 with 0.86 µM insulin and was changed every second day. In addition, (A) 0.5 µM PPARγ agonist rosiglitazone (Rosi), 0.5 µM PPARδ agonist L165041, or 20 µM PPARα agonist Wy14643 was added for the indicated time periods; Significant differences relative to control are indicated by asterisks, ***p<0.001, n = 6. (B) Rosi, <i>n</i>-6 arachidonic acid (ARA<i>n</i>6), <i>n</i>-3 arachidonic acid (ARA<i>n</i>3), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linoleic acid (LA), α-linoleic acid (LNA), oleic acid (Ole), and palmitic acid (Palm) dissolved in DMSO were added at the indicated concentrations from day 3 to day 9. GPDH activities were determined on day 14. In panel B, data presented were normalized to Rosi treated cells. Significant differences relative to Rosi are indicated by asterisks, ***p<0.001, n = 6. C) Two-day post-confluent hMADS cells were maintained for 3 days in serum-free DMEM/Ham's F12 medium in the presence of 1 µM Dex and 0.86 µM insulin supplemented with either 0.5 mM IBMX or various fatty acids as indicated. From day 3 to day 9, 0.5 µM Rosi was then added and GPDH activities were determined on day 14. Significant differences relative to IBMX-treated cells are indicated by asterisks, **p<0.01, ***p<0.001, n = 6.</p