Interactions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesis
<p><b>Background:</b> Neurogenesis requires orchestration of energy status and metabolism. The AMPK pathway which feeds into the mTOR pathway, is activated upon a reduced ratio of ATP to AMP, and through altering metabolic balance, can restore ATP levels (Xiao et al., 2011). Glucos...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2023
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| _version_ | 1826313475689807872 |
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| author | Baig, S |
| author2 | Cader, Z |
| author_facet | Cader, Z Baig, S |
| author_sort | Baig, S |
| collection | OXFORD |
| description | <p><b>Background:</b> Neurogenesis requires orchestration of energy status and metabolism. The AMPK pathway which feeds into the mTOR pathway, is activated upon a reduced ratio of ATP to AMP, and through altering metabolic balance, can restore ATP levels (Xiao et al., 2011). Glucose has been reported to have potent effects on AMPK and mTOR, with TSC2 as a key intermediary (Huynh et al., 2023). However, previous experiments have largely been undertaken in immortalised cell lines and the physiological relevance is therefore unclear. </p>
<p><b>Aims:</b> The goal was to understand how glucose supply affects energy status and mTOR pathway activity during early neurogenesis. </p>
<p><b>Methods:</b> Isogenic human embryonic stem (ES) cells with or without a TSC2 gene deletion were differentiated into neuroepithelial sheets as first described by Shi et al. (2012) under high glucose [25mM] or physiological glucose [5mM] conditions for 12 days. ATP levels, AMPK activation and mTOR activation were measured over differentiation time.</p>
<p><b>Results:</b> ATP levels significantly increased over time, irrespective of media glucose levels in wildtype ES cells differentiated to neuroepithelium (p<0.001). In contrast, AMPK activity was stable regardless of glucose supply or time-point. mTORC1 activity significantly reduced as differentiation progressed, peaking at Day 1 in 5mM glucose and at Day 6 in 25mM glucose (p<0.0001), Metformin, an AMPK activator, had no effect on ATP levels or AMPK activation in either condition.</p>
<p>ATP levels decreased in TSC2 -/- compared to TSC2 +/+ cells in both glucose conditions. mTORC1 activity in TSC2 -/- significantly increased as differentiation progressed (p<0.0001), where activity was significantly reduced in hyperglycaemic conditions. Metformin reversed the very high AMPK activity at Day 12 in TSC2-/- cells, while having no effect on mTORC1 activity at any time-point.</p>
<p><b>Findings:</b> Wildtype cells appeared to have stable ATP levels and AMPK activity irrespective of altered glucose supply. However, a shift in mTORC1 dynamics during early neuronal differentiation was evident. Cells without TSC2 present with dysregulated AMPK in physiological relevant conditions and significantly suppressed mTORC1 activity when cells are in hyperglycaemic conditions. The lack of effect of metformin may represent different pathways which are employed to generate ATP in the early stages of neurogenesis.</p> |
| first_indexed | 2024-09-25T04:14:02Z |
| format | Thesis |
| id | oxford-uuid:81d6d503-512b-4d01-99d4-79ee6f81c8ad |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:14:02Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:81d6d503-512b-4d01-99d4-79ee6f81c8ad2024-07-12T11:29:59ZInteractions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesisThesishttp://purl.org/coar/resource_type/c_bdccuuid:81d6d503-512b-4d01-99d4-79ee6f81c8adNeurodevelopmentEnglishHyrax Deposit2023Baig, SCader, ZSingh, T<p><b>Background:</b> Neurogenesis requires orchestration of energy status and metabolism. The AMPK pathway which feeds into the mTOR pathway, is activated upon a reduced ratio of ATP to AMP, and through altering metabolic balance, can restore ATP levels (Xiao et al., 2011). Glucose has been reported to have potent effects on AMPK and mTOR, with TSC2 as a key intermediary (Huynh et al., 2023). However, previous experiments have largely been undertaken in immortalised cell lines and the physiological relevance is therefore unclear. </p> <p><b>Aims:</b> The goal was to understand how glucose supply affects energy status and mTOR pathway activity during early neurogenesis. </p> <p><b>Methods:</b> Isogenic human embryonic stem (ES) cells with or without a TSC2 gene deletion were differentiated into neuroepithelial sheets as first described by Shi et al. (2012) under high glucose [25mM] or physiological glucose [5mM] conditions for 12 days. ATP levels, AMPK activation and mTOR activation were measured over differentiation time.</p> <p><b>Results:</b> ATP levels significantly increased over time, irrespective of media glucose levels in wildtype ES cells differentiated to neuroepithelium (p<0.001). In contrast, AMPK activity was stable regardless of glucose supply or time-point. mTORC1 activity significantly reduced as differentiation progressed, peaking at Day 1 in 5mM glucose and at Day 6 in 25mM glucose (p<0.0001), Metformin, an AMPK activator, had no effect on ATP levels or AMPK activation in either condition.</p> <p>ATP levels decreased in TSC2 -/- compared to TSC2 +/+ cells in both glucose conditions. mTORC1 activity in TSC2 -/- significantly increased as differentiation progressed (p<0.0001), where activity was significantly reduced in hyperglycaemic conditions. Metformin reversed the very high AMPK activity at Day 12 in TSC2-/- cells, while having no effect on mTORC1 activity at any time-point.</p> <p><b>Findings:</b> Wildtype cells appeared to have stable ATP levels and AMPK activity irrespective of altered glucose supply. However, a shift in mTORC1 dynamics during early neuronal differentiation was evident. Cells without TSC2 present with dysregulated AMPK in physiological relevant conditions and significantly suppressed mTORC1 activity when cells are in hyperglycaemic conditions. The lack of effect of metformin may represent different pathways which are employed to generate ATP in the early stages of neurogenesis.</p> |
| spellingShingle | Neurodevelopment Baig, S Interactions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesis |
| title | Interactions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesis |
| title_full | Interactions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesis |
| title_fullStr | Interactions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesis |
| title_full_unstemmed | Interactions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesis |
| title_short | Interactions of glucose supply, AMPK and the mTOR pathway during early in vitro human neurogenesis |
| title_sort | interactions of glucose supply ampk and the mtor pathway during early in vitro human neurogenesis |
| topic | Neurodevelopment |
| work_keys_str_mv | AT baigs interactionsofglucosesupplyampkandthemtorpathwayduringearlyinvitrohumanneurogenesis |