Author ORCID Identifier

https://orcid.org/0000-0002-2821-9069

Date Available

10-16-2022

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Neuroscience

First Advisor

Bret N. Smith

Second Advisor

Ramon C. Sun

Abstract

Diabetes is a complex metabolic disorder, of which high blood glucose concentration is the primary hallmark. Type I diabetes mellitus (T1DM) is characterized by the lack of insulin production, due to a poorly understood autoinflammatory cascade. In the words of historian Barnett “Diabetes may no longer be a death sentence, but for more and more people in the 21st century, it will become a life sentence”, making it the focal point of many research groups. It is estimated that around 20 million individuals worldwide live with T1DM.

Effects of long-term chronically elevated blood glucose are not only seen in micro/macro-vascular diseases, retinopathy, peripheral neuropathy, and liver disease but also in the brain as people with T1DM show decreased mental speed and flexibility. Despite these clinical observations, the brain’s role in hyperglycemia remains to be elucidated and could be key to identifying potential insulin-independent interventions to alleviate these effects.

In recent years, insulin-independent mechanisms involved in glucose homeostasis have been discovered, most notably the brain’s capacity to regulate blood glucose. The brainstem dorsal vagal complex (DVC) is the main neuronal center responsible for parasympathetic visceral regulation and has been identified as a microcircuit that is important for the regulation of blood glucose. The present work, utilizing a designer receptor exclusively activated by the designer drug (DREADDs) system to selectively activate GABA neurons within hindbrain circuitry in vivo, demonstrates hindbrain inhibitory microcircuitry as a key mediator of whole-body glucose levels, indicating the role of the parasympathetic nervous system.

Neuronal function is affected by the brain metabolome, especially as glucose metabolism is highly heterogeneous among brain regions. To accurately capture physiological brain metabolome we developed a method utilizing a high-power focused microwave to euthanize animals, and fix and preserve metabolites. To understand how hyperglycemia modulates the central carbon metabolism of several brain regions (neocortex, hippocampus, and dorsal vagal complex) we employed gas chromatography-mass spectroscopy. Utilizing untargeted metabolomics we found that glucose concentration was significantly elevated across all regions but glycogen and glucose-6-phosphate remained unchanged with hyperglycemia only in DVC. Interestingly pyruvate and lactate were unchanged across all regions indicating that hyperglycemia does not affect anaerobic cellular respiration. Intermediates of the tricarboxylic acid (TCA) malate and fumarate are significantly decreased with hyperglycemia only in DVC, suggesting that hyperglycemia in DVC preferentially affects TCA cycle. Furthermore, we observed a significant decrease in glutamate across all regions while glutamine and GABA were unchanged, suggesting neurotransmitter regulation disturbance. Stable isotopic tracing of uniformly labeled 13C6 glucose was employed to assess carbon flux in different brain regions perturbed by T1DM to understand its effect on glycolysis, tricarboxylic acid cycle, and neurotransmitter synthesis. We found that hyperglycemia results in metabolic reprogramming with a significant decrease in glucose utilization and we demonstrated decrease in immunofluorescent labeling of GLUT2, a neuronal glucose transporter, as well as enzymes pyruvate dehydrogenase and pyruvate carboxylase, responsible for anaplerosis of TCA cycle intermediates, indicating glucose hypometabolism phenotype.

This work is the first of its kind to demonstrate the effects of hyperglycemia not on the brain as a whole but rather on specific regions; neocortex, hippocampus and DVC. We shown heterogeneous effects of hyperglycemia on the central carbon metabolism pathways in the brain, where TCA cycle and neurotransmitter regulation are selectively affected in the DVC. Collectively, these data demonstrate that peripheral hyperglycemia results in glucose hypometabolism in the brain and will serve as a starting point in understanding the brain’s metabolic adaptations during hyperglycemia.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.371

Funding Information

This study was supported by the National Institutes of Health grants R01 AG066653 (R.C.S.) in 2021/22, R01 CA266004 (R.C.S.) in 2021/22, NIDDK R01 DK122811 (BNS) in 2019/2020, NINDS R01 NS092552 (BNS) in 2021/22, St Baldrick’s Career Development Award (R.C.S.) in 2021/22, V-Scholar Grant (R.C.S.) in 2021/22, and Rally Foundation Independent Investigator Grant to (R.C.S.) in 2022.

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