RESUMEN
AIMS/HYPOTHESIS: All forms of diabetes result from insufficient functional beta cell mass. Due to the relatively limited expression of several antioxidant enzymes, beta cells are highly vulnerable to pathological levels of reactive oxygen species (ROS), which can lead to the reduction of functional beta cell mass. During early postnatal ages, both human and rodent beta cells go through a burst of proliferation that quickly declines with age. The exact mechanisms that account for neonatal beta cell proliferation are understudied but mitochondrial release of moderated ROS levels has been suggested as one of the main drivers. We previously showed that, apart from its conventional role in protecting beta cells from oxidative stress, the nuclear factor erythroid 2-related factor 2 (NRF2) is also essential for beta cell proliferation. We therefore hypothesised that NRF2, which is activated by ROS, plays an essential role in beta cell proliferation at early postnatal ages. 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 (ßNrf2KO) 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 ßNrf2KO 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 ßNrf2KO mice at early postnatal ages. RNA-seq was performed on 7-day-old ßNrf2KO 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 ßNrf2KO 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 ßNrf2KO 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 ßNrf2KO 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 ßNrf2KO mice (3.4-fold) compared with saline-injected ßNrf2KO mice. Interestingly, RNA-seq of islets isolated from 7-day-old ßNrf2KO mice revealed reduced expression of mitochondrial RNA genes and genes involved in the electron transport chain. Islets isolated from 7-day old ßNrf2KO 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 ßNrf2KO 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 ).
Asunto(s)
Células Secretoras de Insulina , Insulinas , Masculino , Humanos , Ratones , Animales , Niño , Recién Nacido , Lactante , Glucemia/metabolismo , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor 2 Relacionado con NF-E2/genética , Animales Recién Nacidos , Biogénesis de Organelos , Células Secretoras de Insulina/metabolismo , Glucosa/metabolismo , Oxidación-Reducción , ADN Mitocondrial/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
The Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor (TF) that is characterized by two major splice isoforms (α and ß). In acute hyperglycemia, both ChREBP isoforms regulate adaptive ß-expansion; however, during chronic hyperglycemia and glucolipotoxicity, ChREBPß expression surges, leading to ß-cell dedifferentiation and death. 14-3-3 binding to ChREBPα results in its cytoplasmic retention and concomitant suppression of transcriptional activity, suggesting that small molecule-mediated stabilization of this protein-protein interaction (PPI) via molecular glues may represent an attractive entry for the treatment of metabolic disease. Here, we show that structure-based optimizations of a molecular glue tool compound led not only to more potent ChREBPα/14-3-3 PPI stabilizers but also for the first time cellular active compounds. In primary human ß-cells, the most active compound stabilized the ChREBPα/14-3-3 interaction and thus induced cytoplasmic retention of ChREBPα, resulting in highly efficient ß-cell protection from glucolipotoxicity while maintaining ß-cell identity. This study may thus not only provide the basis for the development of a unique class of compounds for the treatment of Type 2 Diabetes but also showcases an alternative 'molecular glue' approach for achieving small molecule control of notoriously difficult targetable TFs.