Diabetic gastroenteropathy includes asymptomatic delayed gastric emptying, dyspepsia with or without mildly
delayed gastric emptying, or gastroparesis, which is characterized by more severe symptoms and delayed
gastric emptying. Diabetic gastroparesis may also result in impaired glucose control, nutritional compromise,
anxiety and depression, and poor quality of life. Diabetic gastroenteropathy is often associated with nitrergic
neuropathy; and loss of nitrergic neurons due in part to repressed transcription of neuronal nitric oxide
synthase (Nos1) has been causally linked to gastroparesis. However, pharmacologically increasing nitric oxide
signaling is not beneficial due to gastric and systemic side effects; and compromised tissue microenvironment
in diabetes may limit the efficacy of transplantation of neurons. We have identified physiological hypoxia
(“physioxia”), which is pronounced in enteric neurons, and hypoxia-inducible factor 1 a (HIF1A), a transcription
factor regulated by molecular oxygen sensor enzymes, as key factors of normal Nos1 expression and NOS1
protein levels. We have also found that HIF1A, in addition to stimulating RNA polymerase 2 pause-release,
increases Nos1 transcription by reconfiguring chromatin loops linking proximal Nos1 regulatory elements to
remote super-enhancers. How DM interferes with Nos1 transcription and epigenetic regulation and whether
these changes are reversible in vivo is unclear. We hypothesize that mitochondrial dysfunction in diabetes
interferes with HIF1A-inducible Nos1 transcription via increased intracellular O2 levels that facilitate the
degradation of HIF1A by prolyl hydroxylase domain enzymes and block HIF1A-mediated transactivation by
upregulating the activity of HIF1AN (Specific Aim 1); via increased ratio of the tricarboxylic acid cycle
metabolites succinate:a-ketoglutarate that inhibits histone and DNA demethylases and upregulates repressive
chromatin (Specific Aim 2); and via reduced ATP synthesis, which impairs chromatin looping and reconfigures
enhancer–promoter interactions (Specific Aim 3). We will study these mechanisms in cultured and freshly
isolated, genetically labeled nitrergic neurons, mouse models and patient samples using epigenomic
techniques including chromatin immunoprecipitation-sequencing and genome-wide chromosome conformation
capture, metabolomics, in-vivo analysis of intracellular O2 levels, RNA sequencing, Western blots, and
confocal microscopy. Mechanistic studies will rely on in-vitro RNA interference, conditional gene deletions in
mice, and CRISPR-Cas9-mediated genome and epigenome editing in cells and mice. Pharmacological studies
will target molecular O2 sensors directly or indirectly including via mitochondrial targets in cultured cells and
mice, where gastric functions will be monitored by noninvasive functional assays. To facilitate translation of our
findings, we will validate key observations in human tissues and attempt to restore NOS1 levels and gastric
functions in mice using drugs with proven efficacy in humans. Linking diabetes-associated mitochondrial
dysfunction, hypoxic signaling and gastric functions will change how we think about diabetic gastroenteropathy.