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 ? (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:?-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.
Gastroparesis in patients with diabetes mellitus lacks curative therapy and represents a significant health care burden. Reduced expression of the enzyme producing the inhibitory neurotransmitter nitric oxide in gastric neurons is a pathogenetically significant alteration in gastroparesis, but pharmacological modulation of nitric oxide signaling has not been beneficial due to side effects. This project will determine how impaired mitochondrial metabolism in diabetes and consequent dysregulation of gene transcription can be reversed to restore nitric oxide-dependent gastric functions using drugs already approved for use in humans.
