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Title: Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation

Abstract

Objectives: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells, however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. Methods: To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors (Oct4, Sox2, Klf4, cMyc). We performed pluripotency characterization and directed differentiation of control and IR-KO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm) and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IR-KO iPSCs. Results: Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3 and cMyc were upregulated while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold respectively in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk, and an increase in the total mTor and Erk proteins in IRKO iPSCs at basal level. Stimulation with leukemia inhibitory factor (LIF) showed a ~33% decrease of phospho-ERK in IRKO iPSCs. Onmore » the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Ppar?, Fabp4, C/ebpa and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1 and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network and several key proteins involved in diverse aspects of growth and embryonic development. Conclusion: We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1 and LIF stimulation in mouse iPSCs and reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.« less

Authors:
 [1]; ORCiD logo [2];  [1];  [1];  [1];  [3]; ORCiD logo [3];  [1]; ORCiD logo [3];  [1]
  1. Harvard Medical School, Boston, MA (United States)
  2. Harvard Medical School, Boston, MA (United States); Univ. of Porto, Porto (Portugal)
  3. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1480130
Alternate Identifier(s):
OSTI ID: 1507758
Report Number(s):
PNNL-SA-136923
Journal ID: ISSN 2212-8778; PII: S2212877818306999
Grant/Contract Number:  
AC05-76RL01830; 3-APF-2017-393-A-N; SFRH/BD/51699/2011; R01 DK67536; R01 DK103215; UC4 DK104167; DP3 DK110844; R01DK077097; R01DK102898; #1-18-PDF-169
Resource Type:
Accepted Manuscript
Journal Name:
Molecular Metabolism
Additional Journal Information:
Journal Volume: 18; Journal ID: ISSN 2212-8778
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Insulin receptor signaling; Pluripotency; Lineage differentiation; Adipocyte; Beta cells; Neurons; Stem cells; Phosphoproteomics; Reprogramming

Citation Formats

Gupta, Manoj K., De Jesus, Dario F., Kahraman, Sevim, Valdez, Ivan A., Shamsi, Farnaz, Yi, Lian, Swensen, Adam C., Tseng, Yu -Hua, Qian, Wei -Jun, and Kulkarni, Rohit N. Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation. United States: N. p., 2018. Web. doi:10.1016/j.molmet.2018.09.003.
Gupta, Manoj K., De Jesus, Dario F., Kahraman, Sevim, Valdez, Ivan A., Shamsi, Farnaz, Yi, Lian, Swensen, Adam C., Tseng, Yu -Hua, Qian, Wei -Jun, & Kulkarni, Rohit N. Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation. United States. doi:10.1016/j.molmet.2018.09.003.
Gupta, Manoj K., De Jesus, Dario F., Kahraman, Sevim, Valdez, Ivan A., Shamsi, Farnaz, Yi, Lian, Swensen, Adam C., Tseng, Yu -Hua, Qian, Wei -Jun, and Kulkarni, Rohit N. Wed . "Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation". United States. doi:10.1016/j.molmet.2018.09.003. https://www.osti.gov/servlets/purl/1480130.
@article{osti_1480130,
title = {Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation},
author = {Gupta, Manoj K. and De Jesus, Dario F. and Kahraman, Sevim and Valdez, Ivan A. and Shamsi, Farnaz and Yi, Lian and Swensen, Adam C. and Tseng, Yu -Hua and Qian, Wei -Jun and Kulkarni, Rohit N.},
abstractNote = {Objectives: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells, however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. Methods: To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors (Oct4, Sox2, Klf4, cMyc). We performed pluripotency characterization and directed differentiation of control and IR-KO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm) and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IR-KO iPSCs. Results: Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3 and cMyc were upregulated while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold respectively in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk, and an increase in the total mTor and Erk proteins in IRKO iPSCs at basal level. Stimulation with leukemia inhibitory factor (LIF) showed a ~33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Ppar?, Fabp4, C/ebpa and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1 and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network and several key proteins involved in diverse aspects of growth and embryonic development. Conclusion: We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1 and LIF stimulation in mouse iPSCs and reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.},
doi = {10.1016/j.molmet.2018.09.003},
journal = {Molecular Metabolism},
number = ,
volume = 18,
place = {United States},
year = {2018},
month = {9}
}

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Works referencing / citing this record:

Insulin and Insulin Receptors in Adipose Tissue Development
journal, February 2019

  • Cignarelli, Angelo; Genchi, Valentina; Perrini, Sebastio
  • International Journal of Molecular Sciences, Vol. 20, Issue 3
  • DOI: 10.3390/ijms20030759