NRF2 is required for neonatal mouse beta cell growth by maintaining redox balance and promoting mitochondrial biogenesis and function.

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 ).

Trichomonas vaginalis adherence phenotypes and extracellular vesicles impact parasite survival in a novel in vivo model of pathogenesis.

Trichomonas vaginalis is a human infective parasite responsible for trichomoniasis-the most common, non-viral, sexually transmitted infection worldwide. T. vaginalis resides exclusively in the urogenital tract of both men and women. In women, T. vaginalis has been found colonizing the cervix and vaginal tract while in men it has been identified in the upper and lower urogenital tract and in secreted fluids such as semen, urethral discharge, urine, and prostatic fluid. Despite the over 270 million cases of trichomoniasis annually worldwide, T. vaginalis continues to be a highly neglected organism and thus poorly studied. Here we have developed a male mouse model for studying T. vaginalis pathogenesis in vivo by delivering parasites into the murine urogenital tract (MUT) via transurethral catheterization. Parasite burden was assessed ex-vivo using a nanoluciferase-based gene expression assay which allowed quantification of parasites pre- and post-inoculation. Using this model and read-out approach, we show that T. vaginalis can be found within MUT tissue up to 72 hrs post-inoculation. Furthermore, we also demonstrate that parasites that exhibit increased parasite adherence in vitro also have higher parasite burden in mice in vivo. These data provide evidence that parasite adherence to host cells aids in parasite persistence in vivo and molecular determinants found to correlate with host cell adherence in vitro are applicable to infection in vivo. Finally, we show that co-inoculation of T. vaginalis extracellular vesicles (TvEVs) and parasites results in higher parasite burden in vivo. These findings confirm our previous in vitro-based predictions that TvEVs assist the parasite in colonizing the host. The establishment of this pathogenesis model for T. vaginalis sets the stage for identifying and examining parasite factors that contribute to and influence infection outcomes.

NRF2 Dysregulation in Mice Leads to Inadequate Beta-Cell Mass Expansion during Pregnancy and Gestational Diabetes.

The late stages of the mammalian pregnancy are accompanied with increased insulin resistance due to the increased glucose demand of the growing fetus. Therefore, as a compensatory response to maintain the maternal normal blood glucose levels, maternal beta-cell mass expands leading to increased insulin release. Defects in beta-cell adaptive expansion during pregnancy can lead to gestational diabetes mellitus (GDM). Although the exact mechanisms that promote GDM are poorly understood, GDM strongly associates with impaired beta-cell proliferation and with increased levels of reactive oxygen species (ROS). Here, we show that NRF2 levels are upregulated in mouse beta-cells at gestation day 15 (GD15) concomitant with increased beta-cell proliferation. Importantly, mice with tamoxifen-induced beta-cell-specific NRF2 deletion display inhibition of beta-cell proliferation, increased beta-cell oxidative stress and elevated levels of beta-cell death at GD15. This results in attenuated beta-cell mass expansion and disturbed glucose homeostasis towards the end of pregnancy. Collectively, these results highlight the importance of NRF2-oxidative stress regulation in beta-cell mass adaptation to pregnancy and suggest NRF2 as a potential therapeutic target for treating GDM.

Regulation of Pdx1 by oxidative stress and Nrf2 in pancreatic beta-cells.

The beta-cell identity gene, pancreatic duodenal homeobox 1 (Pdx1), plays critical roles in many aspects of the life of beta-cells including differentiation, maturation, function, survival and proliferation. High levels of reactive oxygen species (ROS) are extremely toxic to cells and especially to beta-cells due to their relatively low expression of antioxidant enzymes. One of the major mechanisms for beta-cell dysfunction in type-2 diabetes results from oxidative stress-dependent inhibition of PDX1 levels and function. ROS inhibits Pdx1 by reducing Pdx1 mRNA and protein levels, inhibiting PDX1 nuclear localization, and suppressing PDX1 coactivator complexes. The nuclear factor erythroid 2-related factor (Nrf2) antioxidant pathway controls the redox balance and allows the maintenance of high Pdx1 levels. Therefore, pharmacological activation of the Nrf2 pathway may alleviate diabetes by preserving Pdx1 levels.

Maladaptive positive feedback production of ChREBPß underlies glucotoxic ß-cell failure.

Preservation and expansion of ß-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPß) is a nuclear effector of hyperglycemic stress occurring in ß-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPß is necessary for adaptive ß-cell expansion in response to metabolic challenges. Conversely, chronic excessive ß-cell-specific overexpression of ChREBPß results in loss of ß-cell identity, apoptosis, loss of ß-cell mass, and diabetes. Furthermore, ß-cell "glucolipotoxicity" can be prevented by deletion of ChREBPß. Moreover, ChREBPß-mediated cell death is mitigated by overexpression of the alternate CHREBP gene product, ChREBPα, or by activation of the antioxidant Nrf2 pathway in rodent and human ß-cells. We conclude that ChREBPß, whether adaptive or maladaptive, is an important determinant of ß-cell fate and a potential target for the preservation of ß-cell mass in diabetes.

Nrf2 Regulates ß-Cell Mass by Suppressing ß-Cell Death and Promoting ß-Cell Proliferation.

Finding therapies that can protect and expand functional ß-cell mass is a major goal of diabetes research. Here, we generated ß-cell-specific conditional knockout and gain-of-function mouse models and used human islet transplant experiments to examine how manipulating Nrf2 levels affects ß-cell survival, proliferation, and mass. Depletion of Nrf2 in ß-cells results in decreased glucose-stimulated ß-cell proliferation ex vivo and decreased adaptive ß-cell proliferation and ß-cell mass expansion after a high-fat diet in vivo. Nrf2 protects ß-cells from apoptosis after a high-fat diet. Nrf2 loss of function decreases Pdx1 abundance and insulin content. Activating Nrf2 in a ß-cell-specific manner increases ß-cell proliferation and mass and improves glucose tolerance. Human islets transplanted under the kidney capsule of immunocompromised mice and treated systemically with bardoxolone methyl, an Nrf2 activator, display increased ß-cell proliferation. Thus, by managing reactive oxygen species levels, Nrf2 regulates ß-cell mass and is an exciting therapeutic target for expanding and protecting ß-cell mass in diabetes.

Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances ß-cell proliferation and identity and relieves oxidative damage.

OBJECTIVE: Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes ß-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance ß-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). METHODS: Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and ß-cell proliferation and ß-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. RESULTS: EP3 blockade increased ß-cell mass in db/db mice through enhanced ß-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. CONCLUSIONS: The current study suggests that EP3 blockade promotes ß-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage.

Adaptive and maladaptive roles for ChREBP in the liver and pancreatic islets.

Excessive sugar consumption is a contributor to the worldwide epidemic of cardiometabolic disease. Understanding mechanisms by which sugar is sensed and regulates metabolic processes may provide new opportunities to prevent and treat these epidemics. Carbohydrate Responsive-Element Binding Protein (ChREBP) is a sugar-sensing transcription factor that mediates genomic responses to changes in carbohydrate abundance in key metabolic tissues. Carbohydrate metabolites activate the canonical form of ChREBP, ChREBP-alpha, which stimulates production of a potent, constitutively active ChREBP isoform called ChREBP-beta. Carbohydrate metabolites and other metabolic signals may also regulate ChREBP activity via posttranslational modifications including phosphorylation, acetylation, and O-GlcNAcylation that can affect ChREBP's cellular localization, stability, binding to cofactors, and transcriptional activity. In this review, we discuss mechanisms regulating ChREBP activity and highlight phenotypes and controversies in ChREBP gain- and loss-of-function genetic rodent models focused on the liver and pancreatic islets.

Nrf2: The Master and Captain of Beta Cell Fate.

Prolonged hyperglycemia is toxic to pancreatic ß cells, generating excessive reactive oxygen species, defective glucose-stimulated insulin secretion, decreased insulin production, and eventually ß cell death and diabetes. Nrf2 is a master regulator of cellular responses to counteract dangerous levels of oxidative stress. Maintenance of ß cell mass depends on Nrf2 to promote the survival, function, and proliferation of ß cells. Indeed, Nrf2 activation decreases inflammation, increases insulin sensitivity, reduces body weight, and preserves ß cell mass. Therefore, numerous pharmacological activators of Nrf2 are being tested in clinical trials for the treatment of diabetes and diabetic complications. Modulating Nrf2 activity in ß cells is a promising therapeutic approach for the treatment of diabetes.

The many lives of Myc in the pancreatic ß-cell.

Diabetes results from insufficient numbers of functional pancreatic ß-cells. Thus, increasing the number of available functional ß-cells ex vivo for transplantation, or regenerating them in situ in diabetic patients, is a major focus of diabetes research. The transcription factor, Myc, discovered decades ago lies at the nexus of most, if not all, known proliferative pathways. Based on this, many studies in the 1990s and early 2000s explored the potential of harnessing Myc expression to expand ß-cells for diabetes treatment. Nearly all these studies in ß-cells used pathophysiological or supraphysiological levels of Myc and reported enhanced ß-cell death, dedifferentiation, or the formation of insulinomas if cooverexpressed with Bcl-xL, an inhibitor of apoptosis. This obviously reduced the enthusiasm for Myc as a therapeutic target for ß-cell regeneration. However, recent studies indicate that "gentle" induction of Myc expression enhances ß-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for ß-cell regeneration. Furthermore, although it has been known for decades that Myc is induced by glucose in ß-cells, very little is known about how this essential anabolic transcription factor perceives and responds to nutrients and increased insulin demand in vivo. Here we summarize the previous and recent knowledge of Myc in the ß-cell, its potential for ß-cell regeneration, and its physiological importance for neonatal and adaptive ß-cell expansion.

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.

Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite's survival in its host. In order to obtain insight into the mechanism of E. histolytica's adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 µM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.

Entamoeba histolytica adaptation to glucose starvation: a matter of life and death.

Parasites are often challenged by constant changes of the glucose concentration in their different hosts and/or within the different biotopes in the same host. During its life cycle, Entamoeba histolytica, the causative protozoan parasite of human amoebiasis, is exposed to both a glucose-poor environment in the colon and a glucose-rich environment in the liver. High-throughput 'omics' technologies are now widely used to characterize the cell's global response to various stresses and these technologies can survey E. histolytica's global response to fluctuations in glucose concentration in its environment. In this review, we discuss the phenotypic and metabolic responses of E. histolytica to glucose challenges, and compare these responses to those of other protozoan parasites.

Identification of dihydropyrimidine dehydrogenase as a virulence factor essential for the survival of Entamoeba histolytica in glucose-poor environments.

Adaptation to nutritional changes is a key feature for successful survival of a pathogen within its host. The protozoan parasite Entamoeba histolytica normally colonizes the human colon and in rare occasions, this parasite spread to distant organs, such as the liver. E. histolytica obtains most of its energy from the fermentation of glucose into ethanol. In this study, we were intrigued to know how this parasite reacts to changes in glucose availability and we addressed this issue by performing a DNA microarray analysis of gene expression. Results show that parasites that were adapted to growth in absence of glucose increased their virulence and altered the transcription of several genes. One of these genes is the dihydropyrimidine dehydrogenase (DPD), which is involved in degradation of pyrimidines. We showed that this gene is crucial for the parasite's growth when the availability of glucose is limited. These data contribute to our understanding of the parasite's ability to survive in glucose-poor environments and reveal a new role for the DPD enzyme.