Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21.

Type 1 diabetes (T1D) is a metabolic disease resulting from progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the majority of beta cells are lost in T1D, a small subset undergoes senescence, a stress response involving growth arrest, DNA damage response, and activation of a senescence-associated secretory phenotype (SASP). SASP in beta cells of the nonobese diabetic (NOD) mouse model of T1D and primary human islets is regulated at the level of transcription by bromodomain extra-terminal (BET) proteins, but the mechanisms remain unclear. To explore how SASP is transcriptionally regulated in beta cells, we used the NOD beta cell line NIT-1 to model beta cell SASP and identified binding partners of BET protein Brd4 and explored the role of the cyclin-dependent kinase inhibitor p21. Brd4 interacted with a variety of proteins in senescent NIT-1 cells including subunits of the Ino80 chromatin remodeling complex, which was expressed in beta cells during T1D progression in NOD mice and in human beta cells of control, autoantibody-positive, and T1D donors as determined from single-cell RNA-seq data. RNAi knockdown of p21 during senescence in NIT-1 cells did not significantly impact viability or SASP. Taken together, these results suggest that Brd4 interacts with several protein partners during senescence in NIT-1 cells, some of which may play roles in SASP gene activation and that p21 is dispensable for the SASP in this beta cell model.

Stress-induced ß cell early senescence confers protection against type 1 diabetes.

During the progression of type 1 diabetes (T1D), ß cells are exposed to significant stress and, therefore, require adaptive responses to survive. The adaptive mechanisms that can preserve ß cell function and survival in the face of autoimmunity remain unclear. Here, we show that the deletion of the unfolded protein response (UPR) genes Atf6α or Ire1α in ß cells of non-obese diabetic (NOD) mice prior to insulitis generates a p21-driven early senescence phenotype and alters the ß cell secretome that significantly enhances the leukemia inhibitory factor-mediated recruitment of M2 macrophages to islets. Consequently, M2 macrophages promote anti-inflammatory responses and immune surveillance that cause the resolution of islet inflammation, the removal of terminally senesced ß cells, the reduction of ß cell apoptosis, and protection against T1D. We further demonstrate that the p21-mediated early senescence signature is conserved in the residual ß cells of T1D patients. Our findings reveal a previously unrecognized link between ß cell UPR and senescence that, if leveraged, may represent a novel preventive strategy for T1D.

Alpha cell dysfunction in type 1 diabetes is independent of a senescence program.

Type 1 Diabetes (T1D) is caused by insulin deficiency, due to progressive autoimmune destruction of pancreatic ß cells. Glucagon-secreting α cells become dysfunctional in T1D and contribute to pathophysiology, however, the mechanisms involved are unclear. While the majority of ß cells are destroyed in T1D, some ß cells escape this fate and become senescent but whether α cell dysfunction involves a senescence program has not been explored. Here we addressed the question of whether α cells become senescent during the natural history of T1D in the non-obese diabetic (NOD) mouse model and humans. NOD mice had several distinct subpopulations of α cells, but none were defined by markers of senescence at the transcriptional or protein level. Similarly, α cells of human T1D donors did not express senescence markers. Despite the lack of senescence in α cells in vivo, using a human islet culture model, we observed that DNA damage-induced senescence led to alterations in islet glucagon secretion, which could be rescued by inhibiting the senescence-associated secretory phenotype (SASP). Together our results suggest that α cell dysfunction in T1D is not due to activation of a senescence program, however, senescent ß cell accumulation in the islet microenvironment may have a negative effect on α cell function.

DNA damage to ß cells in culture recapitulates features of senescent ß cells that accumulate in type 1 diabetes.

OBJECTIVE: Type 1 Diabetes (T1D) is characterized by progressive loss of insulin-producing pancreatic ß cells as a result of autoimmune destruction. In addition to ß cell death, recent work has shown that subpopulations of ß cells acquire dysfunction during T1D. We previously reported that ß cells undergoing a DNA damage response (DDR) and senescence accumulate during the pathogenesis of T1D. However, the question of how senescence develops in ß cells has not been investigated. METHODS: Here, we tested the hypothesis that unrepaired DNA damage in the context of genetic susceptibility triggers ß cell senescence using culture models including the mouse NIT1 ß cell line derived from the T1D-susceptible nonobese diabetic (NOD) strain, human donor islets and EndoC ß cells. DNA damage was chemically induced using etoposide or bleomycin and cells or islets were analyzed by a combination of molecular assays for senescence phenotypes including Western blotting, qRT-PCR, Luminex assays, flow cytometry and histochemical staining. RNA-seq was carried out to profile global transcriptomic changes in human islets undergoing DDR and senescence. Insulin ELISAs were used to quantify glucose-stimulated insulin secretion from chemically-induced senescent human islets, EndoC ß cells and mouse ß cell lines in culture. RESULTS: Sub-lethal DNA damage in NIT1 cells led to several classical hallmarks of senescence including sustained DDR activation, growth arrest, enlarged flattened morphology and a senescence-associated secretory phenotype (SASP) resembling what occurs in primary ß cells during T1D in NOD mice. These phenotypes differed between NIT1 cells and the MIN6 ß cell line derived from a non-T1D susceptible mouse strain. RNA-seq analysis of DNA damage-induced senescence in human islets from two different donors revealed a p53 transcriptional program and upregulation of prosurvival and SASP genes, with inter-donor variability in this response. Inter-donor variability in human islets was also apparent in the extent of persistent DDR activation and SASP at the protein level. Notably, chemically induced DNA damage also led to DDR activation and senescent phenotypes in EndoC-ßH5 human ß cells, confirming that this response can occur directly in a human ß cell line. Finally, DNA damage led to different effects on glucose-stimulated insulin secretion in mouse ß cell lines as compared with human islets and EndoC ß cells. CONCLUSIONS: Taken together, these findings suggest that some of the phenotypes of senescent ß cells that accumulate during the development of T1D in the NOD mouse and humans can be modeled by chemically induced DNA damage to mouse ß cell lines, human islets and EndoC ß cells in culture. The differences between ß cells from different mouse strains and different human islet donors and EndoC ß cells highlights species differences and the role for genetic background in modifying the ß cell response to DNA damage and its effects on insulin secretion. These culture models will be useful tools to understand some of the mechanisms of ß cell senescence in T1D.

Beta Cell Therapies for Preventing Type 1 Diabetes: From Bench to Bedside.

Type 1 diabetes (T1D) is a chronic metabolic disease characterized by insulin deficiency, generally resulting from progressive autoimmune-mediated destruction of pancreatic beta cells. While the phenomenon of beta cell autoimmunity continues to be an active area of investigation, recent evidence suggests that beta cell stress responses are also important contributors to disease onset. Here we review the pathways driving different kinds of beta cell dysfunction and their respective therapeutic targets in the prevention of T1D. We discuss opportunities and important open questions around the effectiveness of beta cell therapies and challenges for clinical utility. We further evaluate ways in which beta cell drug therapy could be combined with immunotherapy for preventing T1D in light of our growing appreciation of disease heterogeneity and patient endotypes. Ultimately, the emergence of pharmacologic beta cell therapies for T1D have armed us with new tools and closing the knowledge gaps in T1D etiology will be essential for maximizing the potential of these approaches.

Maternal resveratrol administration protects against gestational diabetes-induced glucose intolerance and islet dysfunction in the rat offspring.

KEY POINTS: Maternal resveratrol (RESV) administration in gestational diabetes (GDM) restored normoglycaemia and insulin secretion. GDM-induced obesity was prevented in male GDM+RESV offspring but not in females. GDM+RESV offspring exhibited improved glucose tolerance and insulin sensitivity. GDM+RESV restored hepatic glucose homeostasis in offspring. Glucose-stimulated insulin secretion was enhanced in GDM+RESV offspring. ABSTRACT: Gestational diabetes (GDM), the most common complication of pregnancy, is associated with adverse metabolic health outcomes in offspring. Using a rat model of diet-induced GDM, we investigated whether maternal resveratrol (RESV) supplementation (147 mg kg-1  day-1 ) in the third week of pregnancy could improve maternal glycaemia and protect the offspring from developing metabolic dysfunction. Female Sprague-Dawley rats consumed a high-fat and sucrose (HFS) diet to induce GDM. Lean controls consumed a low-fat (LF) diet. In the third trimester, when maternal hyperglycaemia was observed, the HFS diet was supplemented with RESV. At weaning, offspring were randomly assigned a LF or HFS diet until 15 weeks of age. In pregnant dams, RESV restored glucose tolerance, normoglycaemia and improved insulin secretion. At 15 weeks of age, GDM+RESV-HFS male offspring were less obese than the GDM-HFS offspring. By contrast, the female GDM+RESV-HFS offspring were similarly as obese as the GDM-HFS group. Hepatic steatosis, insulin resistance, glucose intolerance and dysregulated gluconeogenesis were observed in the male GDM offspring and were attenuated in the offspring of GDM+RESV dams. The dysregulation of several metabolic genes (e.g. ppara, lpl, pepck and g6p) in the livers of GDM offspring was attenuated in the GDM+RESV offspring group. Glucose stimulated insulin secretion was also improved in the islets from offspring of GDM+RESV dams. Thus, maternal RESV supplementation during the third trimester of pregnancy and lactation induced several beneficial metabolic health outcomes for both mothers and offspring. Therefore, RESV could be an alternative to current GDM treatments.

Therapies for gestational diabetes and their implications for maternal and offspring health: Evidence from human and animal studies.

Obesity prior to and during pregnancy is associated with an increased risk of complications during pregnancy. One of the most common complications of pregnancy is gestational diabetes mellitus (GDM), a condition characterized by hyperglycemia and insulin resistance that is diagnosed in the third trimester of pregnancy. GDM predisposes both mothers and their children to increased obesity and cardiometabolic disorders, namely type 2 diabetes and cardiovascular disease. Current treatments include lifestyle changes and insulin injections, but oral anti-diabetic drugs such as metformin and glyburide are increasingly prescribed as they do not require injections. However, the long-term implications of therapies for diabetes during pregnancy on mothers and their offspring are not fully understood. In this review, we describe current treatments for GDM, including the first line lifestyle interventions such as exercise as well as insulin, glyburides and metformin. We also review selected natural health products that are sometimes used by individuals during pregnancy that could also be an effective therapeutic in pregnancies characterized by obesity or GDM. We focus on both the short- and long-term effects of treatments on the health of mothers and their offspring. We review the current literature from clinical research and animal studies.

Maternal Macronutrient Consumption and the Developmental Origins of Metabolic Disease in the Offspring.

Recent research aimed at understanding the rise in obesity and cardiometabolic disease in children suggests that suboptimal maternal nutrition conditions organ systems and physiological responses in the offspring contributing to disease development. Understanding the mechanisms by which the macronutrient composition of the maternal diet during pregnancy or lactation affects health outcomes in the offspring may lead to new maternal nutrition recommendations, disease prevention strategies and therapies that reduce the increasing incidence of cardiometabolic disease in children. Recent mechanistic animal model research has identified how excess fats and sugars in the maternal diet alter offspring glucose tolerance, insulin signaling and metabolism. Maternal nutrition appears to influence epigenetic alterations in the offspring and the programming of gene expression in key metabolic pathways. This review is focused on experimental studies in animal models that have investigated mechanisms of how maternal consumption of macronutrients affects cardiometabolic disease development in the offspring. Future research using "-omic" technologies is essential to elucidate the mechanisms of how altered maternal macronutrient consumption influences the development of disease in the offspring.

The global regulator ANR is essential for Pseudomonas chlororaphis strain PA23 biocontrol.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungus Sclerotinia sclerotiorum. The focus of the current study was to elucidate the role of the transcriptional regulator ANR in the biocontrol capabilities of this bacterium. An anr mutant was created, PA23anr, that was devoid antifungal activity. In other pseudomonads, ANR is essential for regulating HCN production. Characterization of PA23anr revealed that, in addition to HCN, ANR controls phenazine (PHZ), pyrrolnitrin (PRN), protease and autoinducer (AHL) signal molecule production. In gene expression studies, hcnA, phzA, prnA and phzI were found to be downregulated, consistent with our endproduct analysis. Because the phenotype of PA23anr closely resembles that of quorum sensing (QS)-deficient strains, we explored whether there is a connection between ANR and the PhzRI QS system. Both phzI and phzR are positively regulated by ANR, whereas PhzR represses anr transcription. Complementation of PA23anr with pUCP-phzR, C6-HSL or both yielded no change in phenotype. Conversely, PA23phzR harbouring pUCP23-anr exhibited partial-to-full restoration of antifungal activity, HCN, PRN and AHL production together with hcnA, prnA, phzI and rpoS expression. PHZ and protease production remained unchanged indicating that ANR can complement the QS-deficient phenotype with respect to some but not all traits. Our experiments were conducted at atmospheric O2 levels underscoring the fact that ANR has a profound effect on PA23 physiology under aerobic conditions.