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Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

Chen RJ, Zhang G, Garfield SH, Shi YJ, Chen KG, Robey PG, Leapman RD - PLoS ONE (2015)

Bottom Line: Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway.Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis.The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, United States of America.

ABSTRACT
Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

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Related in: MedlinePlus

Growth, differentiation, and glycogen synthesis in human embryonic stem cells (hESCs).A hypothetical model is presented to elucidate major signaling pathways that are associated with glycogen synthase kinase 3 (GSK-3) and glycogen synthesis. (A) In this model, glucose transporter-mediated uptake of glucose is activated by an insulin-receptor signaling pathway. (B) Glucose takes part in aerobic glycolysis in the cytoplasm and oxidative phosphorylation in mitochondria to produce energy for hESC proliferation and self-renewal. Presumably, excessive glucose is converted to glycogen by activated glycogen synthase (GSa) upon stress and differentiation signaling to enhance hPSC survival. Glycogen can be decomposed in the presence of phosphorylated glycogen phorsphoylase (pGP) whenever necessary. (C) The insulin signaling pathway also activates the PI3K-AKT pathway, which phosphorylates GSK-3. The GSK-3 phosphorylation leads to its inactivation and subsequently inhibits the phosphorylation of glycogen synthase (GS). Thus, activation of the PI3K-AKT pathway increases glycogen synthesis. (D) The mechanism of BMP-4-induced glycogen body formation is likely through the inhibition of GSK-3 by the putative Smad pathways. (E) The mechanism by which the GSK3i CHIR modulates the synthesis of glycogen is likely through the inhibition of GSK-3 activity, thereby altering glycogen synthase activity. (F) Concomitantly, GSK-3 inhibitors (e.g., CHIR99021 and BIO) may promote hPSC differentiation by activation of the β-catenin-WNT pathway. (G) The function of aggregated glycogen bodies is unclear and may be associated with response to extracellular stress and differentiation signals such as BMP-4. (H) Under sustained Oct-4 expression conditions, GSK3i-mediated glycogen accumulation concomitant with Wnt activation and other naïve growth components enhances the transition from the primed pluripotent to the naïve state in hPSCs. The proposed mechanisms in this model supported by this study are color-highlighted. The “?” symbols indicate inconclusive observations. The abbreviations are: 2iL, the naïve pluripotent growth condition that include GSK3i, MEKi, and LIF; 3iL, the naïve pluripotent growth condition that include GSK3i, MEKi, BMP4i, and LIF; AKT, the serine-threonine protein kinase encoded by v-akt murine thymoma viral oncogene homolog; CHIR, CHIR99021; GPi, dephosphorylated glycogen phosphorylase (inactive form); GSa, dephosphorylated glycogen synthase (active form); GSK-3, glycogen synthase kinase 3; pGPa, phosphorylated glycogen phosphorylase (active form); pGSi, phosphorylated glycogen synthase (inactive form); PI3K, the phosphoinositide 3-kinase; and β-cat, β-catenin.
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pone.0142554.g009: Growth, differentiation, and glycogen synthesis in human embryonic stem cells (hESCs).A hypothetical model is presented to elucidate major signaling pathways that are associated with glycogen synthase kinase 3 (GSK-3) and glycogen synthesis. (A) In this model, glucose transporter-mediated uptake of glucose is activated by an insulin-receptor signaling pathway. (B) Glucose takes part in aerobic glycolysis in the cytoplasm and oxidative phosphorylation in mitochondria to produce energy for hESC proliferation and self-renewal. Presumably, excessive glucose is converted to glycogen by activated glycogen synthase (GSa) upon stress and differentiation signaling to enhance hPSC survival. Glycogen can be decomposed in the presence of phosphorylated glycogen phorsphoylase (pGP) whenever necessary. (C) The insulin signaling pathway also activates the PI3K-AKT pathway, which phosphorylates GSK-3. The GSK-3 phosphorylation leads to its inactivation and subsequently inhibits the phosphorylation of glycogen synthase (GS). Thus, activation of the PI3K-AKT pathway increases glycogen synthesis. (D) The mechanism of BMP-4-induced glycogen body formation is likely through the inhibition of GSK-3 by the putative Smad pathways. (E) The mechanism by which the GSK3i CHIR modulates the synthesis of glycogen is likely through the inhibition of GSK-3 activity, thereby altering glycogen synthase activity. (F) Concomitantly, GSK-3 inhibitors (e.g., CHIR99021 and BIO) may promote hPSC differentiation by activation of the β-catenin-WNT pathway. (G) The function of aggregated glycogen bodies is unclear and may be associated with response to extracellular stress and differentiation signals such as BMP-4. (H) Under sustained Oct-4 expression conditions, GSK3i-mediated glycogen accumulation concomitant with Wnt activation and other naïve growth components enhances the transition from the primed pluripotent to the naïve state in hPSCs. The proposed mechanisms in this model supported by this study are color-highlighted. The “?” symbols indicate inconclusive observations. The abbreviations are: 2iL, the naïve pluripotent growth condition that include GSK3i, MEKi, and LIF; 3iL, the naïve pluripotent growth condition that include GSK3i, MEKi, BMP4i, and LIF; AKT, the serine-threonine protein kinase encoded by v-akt murine thymoma viral oncogene homolog; CHIR, CHIR99021; GPi, dephosphorylated glycogen phosphorylase (inactive form); GSa, dephosphorylated glycogen synthase (active form); GSK-3, glycogen synthase kinase 3; pGPa, phosphorylated glycogen phosphorylase (active form); pGSi, phosphorylated glycogen synthase (inactive form); PI3K, the phosphoinositide 3-kinase; and β-cat, β-catenin.

Mentions: Furthermore, using naïve human stem cell medium (NHSM) that contains LIF and 2i, Hanna and colleagues were also able to directly convert primed hPSCs to the naïve state [15]. Based on 2i and LIF, several research groups have successfully developed different protocols for direct conversion of primed hPSCs to the naïve state [13, 16–18]. We used a naïve hPSC culture protocol based on the 3iL [13], which was compatible with our current culture platforms. We were able to systematically address glycogen variations under different naïve conditions (e.g., BIO, 2i, 3i, 2iL, and 3iL). Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis (Figs 5–8). It appears that GSK3i is a necessary, but insufficient element, for the regulation of the distinct pluripotent states. However, it is the essential component to trigger the naïve state associated glycogen synthesis. The addition of more naïve components such as MEKi and LIF in the naïve growth protocols might play a role in stabilizing the metabolic state that are associated with the naïve pluripotency (Fig 9).


Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

Chen RJ, Zhang G, Garfield SH, Shi YJ, Chen KG, Robey PG, Leapman RD - PLoS ONE (2015)

Growth, differentiation, and glycogen synthesis in human embryonic stem cells (hESCs).A hypothetical model is presented to elucidate major signaling pathways that are associated with glycogen synthase kinase 3 (GSK-3) and glycogen synthesis. (A) In this model, glucose transporter-mediated uptake of glucose is activated by an insulin-receptor signaling pathway. (B) Glucose takes part in aerobic glycolysis in the cytoplasm and oxidative phosphorylation in mitochondria to produce energy for hESC proliferation and self-renewal. Presumably, excessive glucose is converted to glycogen by activated glycogen synthase (GSa) upon stress and differentiation signaling to enhance hPSC survival. Glycogen can be decomposed in the presence of phosphorylated glycogen phorsphoylase (pGP) whenever necessary. (C) The insulin signaling pathway also activates the PI3K-AKT pathway, which phosphorylates GSK-3. The GSK-3 phosphorylation leads to its inactivation and subsequently inhibits the phosphorylation of glycogen synthase (GS). Thus, activation of the PI3K-AKT pathway increases glycogen synthesis. (D) The mechanism of BMP-4-induced glycogen body formation is likely through the inhibition of GSK-3 by the putative Smad pathways. (E) The mechanism by which the GSK3i CHIR modulates the synthesis of glycogen is likely through the inhibition of GSK-3 activity, thereby altering glycogen synthase activity. (F) Concomitantly, GSK-3 inhibitors (e.g., CHIR99021 and BIO) may promote hPSC differentiation by activation of the β-catenin-WNT pathway. (G) The function of aggregated glycogen bodies is unclear and may be associated with response to extracellular stress and differentiation signals such as BMP-4. (H) Under sustained Oct-4 expression conditions, GSK3i-mediated glycogen accumulation concomitant with Wnt activation and other naïve growth components enhances the transition from the primed pluripotent to the naïve state in hPSCs. The proposed mechanisms in this model supported by this study are color-highlighted. The “?” symbols indicate inconclusive observations. The abbreviations are: 2iL, the naïve pluripotent growth condition that include GSK3i, MEKi, and LIF; 3iL, the naïve pluripotent growth condition that include GSK3i, MEKi, BMP4i, and LIF; AKT, the serine-threonine protein kinase encoded by v-akt murine thymoma viral oncogene homolog; CHIR, CHIR99021; GPi, dephosphorylated glycogen phosphorylase (inactive form); GSa, dephosphorylated glycogen synthase (active form); GSK-3, glycogen synthase kinase 3; pGPa, phosphorylated glycogen phosphorylase (active form); pGSi, phosphorylated glycogen synthase (inactive form); PI3K, the phosphoinositide 3-kinase; and β-cat, β-catenin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643957&req=5

pone.0142554.g009: Growth, differentiation, and glycogen synthesis in human embryonic stem cells (hESCs).A hypothetical model is presented to elucidate major signaling pathways that are associated with glycogen synthase kinase 3 (GSK-3) and glycogen synthesis. (A) In this model, glucose transporter-mediated uptake of glucose is activated by an insulin-receptor signaling pathway. (B) Glucose takes part in aerobic glycolysis in the cytoplasm and oxidative phosphorylation in mitochondria to produce energy for hESC proliferation and self-renewal. Presumably, excessive glucose is converted to glycogen by activated glycogen synthase (GSa) upon stress and differentiation signaling to enhance hPSC survival. Glycogen can be decomposed in the presence of phosphorylated glycogen phorsphoylase (pGP) whenever necessary. (C) The insulin signaling pathway also activates the PI3K-AKT pathway, which phosphorylates GSK-3. The GSK-3 phosphorylation leads to its inactivation and subsequently inhibits the phosphorylation of glycogen synthase (GS). Thus, activation of the PI3K-AKT pathway increases glycogen synthesis. (D) The mechanism of BMP-4-induced glycogen body formation is likely through the inhibition of GSK-3 by the putative Smad pathways. (E) The mechanism by which the GSK3i CHIR modulates the synthesis of glycogen is likely through the inhibition of GSK-3 activity, thereby altering glycogen synthase activity. (F) Concomitantly, GSK-3 inhibitors (e.g., CHIR99021 and BIO) may promote hPSC differentiation by activation of the β-catenin-WNT pathway. (G) The function of aggregated glycogen bodies is unclear and may be associated with response to extracellular stress and differentiation signals such as BMP-4. (H) Under sustained Oct-4 expression conditions, GSK3i-mediated glycogen accumulation concomitant with Wnt activation and other naïve growth components enhances the transition from the primed pluripotent to the naïve state in hPSCs. The proposed mechanisms in this model supported by this study are color-highlighted. The “?” symbols indicate inconclusive observations. The abbreviations are: 2iL, the naïve pluripotent growth condition that include GSK3i, MEKi, and LIF; 3iL, the naïve pluripotent growth condition that include GSK3i, MEKi, BMP4i, and LIF; AKT, the serine-threonine protein kinase encoded by v-akt murine thymoma viral oncogene homolog; CHIR, CHIR99021; GPi, dephosphorylated glycogen phosphorylase (inactive form); GSa, dephosphorylated glycogen synthase (active form); GSK-3, glycogen synthase kinase 3; pGPa, phosphorylated glycogen phosphorylase (active form); pGSi, phosphorylated glycogen synthase (inactive form); PI3K, the phosphoinositide 3-kinase; and β-cat, β-catenin.
Mentions: Furthermore, using naïve human stem cell medium (NHSM) that contains LIF and 2i, Hanna and colleagues were also able to directly convert primed hPSCs to the naïve state [15]. Based on 2i and LIF, several research groups have successfully developed different protocols for direct conversion of primed hPSCs to the naïve state [13, 16–18]. We used a naïve hPSC culture protocol based on the 3iL [13], which was compatible with our current culture platforms. We were able to systematically address glycogen variations under different naïve conditions (e.g., BIO, 2i, 3i, 2iL, and 3iL). Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis (Figs 5–8). It appears that GSK3i is a necessary, but insufficient element, for the regulation of the distinct pluripotent states. However, it is the essential component to trigger the naïve state associated glycogen synthesis. The addition of more naïve components such as MEKi and LIF in the naïve growth protocols might play a role in stabilizing the metabolic state that are associated with the naïve pluripotency (Fig 9).

Bottom Line: Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway.Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis.The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, United States of America.

ABSTRACT
Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

Show MeSH
Related in: MedlinePlus