Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin.
The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully unders...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2020-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0231806 |
| _version_ | 1819134546258427904 |
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| author | Craig A Eyster Satoshi Matsuzaki Maria F Newhardt Jennifer R Giorgione Kenneth M Humphries |
| author_facet | Craig A Eyster Satoshi Matsuzaki Maria F Newhardt Jennifer R Giorgione Kenneth M Humphries |
| author_sort | Craig A Eyster |
| collection | DOAJ |
| description | The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. PKA substrate phosphorylation was decreased in Akita ACMs, as compared to wild type, and unresponsive to the effects of insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator. |
| first_indexed | 2024-12-22T10:04:54Z |
| format | Article |
| id | doaj.art-35efde930bf549ec895ea70dd3926301 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T10:04:54Z |
| publishDate | 2020-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-35efde930bf549ec895ea70dd39263012022-12-21T18:30:00ZengPublic Library of Science (PLoS)PLoS ONE1932-62032020-01-01158e023180610.1371/journal.pone.0231806Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin.Craig A EysterSatoshi MatsuzakiMaria F NewhardtJennifer R GiorgioneKenneth M HumphriesThe cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. PKA substrate phosphorylation was decreased in Akita ACMs, as compared to wild type, and unresponsive to the effects of insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator.https://doi.org/10.1371/journal.pone.0231806 |
| spellingShingle | Craig A Eyster Satoshi Matsuzaki Maria F Newhardt Jennifer R Giorgione Kenneth M Humphries Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin. PLoS ONE |
| title | Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin. |
| title_full | Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin. |
| title_fullStr | Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin. |
| title_full_unstemmed | Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin. |
| title_short | Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin. |
| title_sort | diabetes induced decreases in pka signaling in cardiomyocytes the role of insulin |
| url | https://doi.org/10.1371/journal.pone.0231806 |
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