Exploring Beta Cell NADPH production using Organelle-targeted Apollo-NADP+ Sensors

A major function of beta cells is the production and secretion of insulin to maintain glucose homeostasis. Loss of this function is directly linked to progression of diabetes. The loss of beta cell function is exacerbated by cellular senescence associated with natural aging. Senescence impacts mitochondrial-dependent pyruvate cycling and nicotinamide nucleotide transhydrogenase (NNT) activity, which are important for production of beta cell NADPH to support biomolecule synthesis during cell growth and for the quenching of reactive oxygen species generated during oxidative stress. We hypothesized there exist changes in beta cell NADPH associated with development of beta cell senescence. We previously developed genetically encoded Apollo-NADP+ sensors to track changes in NADPH/NADP+ redox in real time. We targeted Apollo-NADP+ to various organelles to probe whether organelle compartmentalization of NADPH could create disparate impacts on beta cell metabolism. We demonstrated glucose- and glutamine-stimulated NADPH responses in the mitochondria preceded those in the cytoplasm indicating the dominant role of mitochondria in setting beta cell NADPH redox state. We subsequently aimed to develop an Apollo sensor based around pyruvate kinase M2 to allow for simultaneous tracking of NADPH and glycolytic flux. The resulting sensor was responsive to some oxidative treatments such as hydrogen peroxide and diamide, but wholly insensitive to other physiological treatments such as glucose and serine. It is likely the sensitivity of pyruvate kinase M2 to a variety of post-translational modifications made the sensor responses difficult to interpret. Finally, we explored doxorubicin as an inducer of senescence to observe changes to NADPH production during senescence. We found doxorubicin locked cells into a late G1 state consistent with induction of senescence. Senescent cells appeared to have increased glutamine-stimulated NADPH responses, consistent with reports of senescent cells being dependent on glutamine for survival. Preliminary data suggest the increased NADPH response may be due to an increased proportion of cells in G0/G1. Future work aims to identify differences in NADPH metabolism between quiescent and senescent beta cells. Overall, this thesis developed organelle targeted tools to enable simultaneous study of mitochondrial-driven NADPH responses simultaneously with other sensors.

Department dc:contributor.department

Biomedical Engineering

Year dc:date.issued

2023

Author dc:creator

• Chang, Huntley Hung Chih

Advisor dc:contributor.advisor

• Rocheleau, Jonathan V

• Fluorescent protein
• NADPH redox
• Protein engineering
• Sensors

Handle dc:identifier.uri

http://hdl.handle.net/1807/129775

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/129775