Conclusions/interpretation
Our study highlights NRF2 as an essential transcription factor for maintaining neonatal redox balance, mitochondrial biogenesis and function and beta cell growth, and for preserving functional beta cell mass in adulthood under metabolic stress. Data availability: Sequencing data are available in the NCBI Gene Expression Omnibus, accession number GSE242718 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242718 ).
Methods
Beta cell NRF2 levels and beta cell proliferation were measured in pancreatic sections from non-diabetic human cadaveric donors at different postnatal ages, childhood and adulthood. Pancreatic sections from 1-, 7-, 14- and 28-day-old beta cell-specific Nrf2 (also known as Nfe2l2)-knockout mice (βNrf2KO) or control (Nrf2lox/lox) mice were assessed for beta cell NRF2 levels, beta cell proliferation, beta cell oxidative stress, beta cell death, nuclear beta cell pancreatic duodenal homeobox protein 1 (PDX1) levels and beta cell mass. Seven-day-old βNrf2KO and Nrf2lox/lox mice were injected daily with N-acetylcysteine (NAC) or saline (154 mmol/l NaCl) to explore the potential contribution of oxidative stress to the phenotypes seen in βNrf2KO mice at early postnatal ages. RNA-seq was performed on 7-day-old βNrf2KO and Nrf2lox/lox mice to investigate the mechanisms by which NRF2 stimulates beta cell proliferation at early postnatal ages. Mitochondrial biogenesis and function were determined using dispersed islets from 7-day-old βNrf2KO and Nrf2lox/lox mice by measuring MitoTracker intensity, mtDNA/gDNA ratio and ATP/ADP ratio. To study the effect of neonatal beta cell-specific Nrf2 deletion on glucose homeostasis in adulthood, blood glucose, plasma insulin and insulin secretion were determined and a GTT was performed on 3-month-old βNrf2KO and Nrf2lox/lox mice fed on regular diet (RD) or high-fat diet (HFD).
Results
The expression of the master antioxidant regulator NRF2 was increased at early postnatal ages in both human (1 day to 19 months old, 31%) and mouse (7 days old, 57%) beta cells, and gradually declined with age (8% in adult humans, 3.77% in adult mice). A significant correlation (R2=0.568; p=0.001) was found between beta cell proliferation and NRF2 levels in human beta cells. Seven-day-old βNrf2KO mice showed reduced beta cell proliferation (by 65%), beta cell nuclear PDX1 levels (by 23%) and beta cell mass (by 67%), and increased beta cell oxidative stress (threefold) and beta cell death compared with Nrf2lox/lox control mice. NAC injections increased beta cell proliferation in 7-day-old βNrf2KO mice (3.4-fold) compared with saline-injected βNrf2KO mice. Interestingly, RNA-seq of islets isolated from 7-day-old βNrf2KO mice revealed reduced expression of mitochondrial RNA genes and genes involved in the electron transport chain. Islets isolated from 7-day old βNrf2KO mice presented reduced MitoTracker intensity (by 47%), mtDNA/gDNA ratio (by 75%) and ATP/ADP ratio (by 68%) compared with islets from Nrf2lox/lox littermates. Lastly, HFD-fed 3-month-old βNrf2KO male mice displayed a significant reduction in beta cell mass (by 35%), a mild increase in non-fasting blood glucose (1.2-fold), decreased plasma insulin (by 14%), and reduced glucose tolerance (1.3-fold) compared with HFD-fed Nrf2lox/lox mice. Conclusions/interpretation: Our study highlights NRF2 as an essential transcription factor for maintaining neonatal redox balance, mitochondrial biogenesis and function and beta cell growth, and for preserving functional beta cell mass in adulthood under metabolic stress. Data availability: Sequencing data are available in the NCBI Gene Expression Omnibus, accession number GSE242718 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242718 ).