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Aims/hypothesis Beta cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialised gene expression programme that is established during development and then sustained, with limited flexibility, in terminally differentiated cells. Dysregulation of this programme is seen in type 2 diabetes but mechanisms that preserve gene expression or underlie its dysregulation in mature cells are not well resolved. This study investigated whether methylation of histone H3 lysine 4 (H3K4), a marker of gene promoters with unresolved functional importance, is necessary for the maintenance of mature beta cell function. Methods Beta cell function, gene expression and chromatin modifications were analysed in conditional Dpy30 knockout mice, in which H3K4 methyltransferase activity is impaired, and in a mouse model of diabetes. Results H3K4 methylation maintains expression of genes that are important for insulin biosynthesis and glucose responsiveness. Deficient methylation of H3K4 leads to a less active and more repressed epigenome profile that locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We further show that H3K4 trimethylation (H3K4me3) is reorganised in islets from the Lepr db/db mouse model of diabetes in favour of weakly active and disallowed genes at the expense of terminal beta cell markers with broad H3K4me3 peaks. Conclusions/interpretation Sustained methylation of H3K4 is critical for the maintenance of beta cell function. Redistribution of H3K4me3 is linked to gene expression changes that are implicated in diabetes pathology. Graphical abstract

ER stress and apoptosis contribute to the loss of pancreatic β-cells under pro-diabetic conditions of glucolipotoxicity. Although activation of canonical intrinsic apoptosis is known to require pro-apoptotic Bcl-2 family proteins Bax and Bak, their individual and combined involvement in glucolipotoxic β-cell death are not known. It has also remained an open question if Bax and Bak in β-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined β-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic β-cell death in vitro occurs by both non-apoptotic and apoptotic mechanisms, and the apoptosis could be triggered by either Bax or Bak alone. In contrast, they had non-redundant roles in mediating staurosporine-induced apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated β-cell Ca 2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that combined deletion of Bax and Bak amplified the unfolded protein response in islets during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings shed new light on roles of the core apoptosis machinery in β-cell survival and stress signals of importance for the pathobiology of diabetes.

Diabetes onset is accompanied with β-cell deficiency, and thus restoring functional β-cell mass is a promising therapy for those with diabetes. However, the regulatory mechanisms controlling β-cell mass are not fully understood. Previously, we demonstrated that the transcription factor SOX4 is required for β-cell proliferation in the prediabetic state. To elucidate potential mechanisms by which SOX4 regulates β-cell mass, we performed RNA sequencing (RNA-seq) using pancreatic β-cell specific SOX4 knockout mice (βSOX4 KO). The RNA-seq revealed decreased expression of GRK5, a known type 2 diabetes susceptibility gene whose association with diabetes has not been fully elucidated. Therefore, we aimed to clarify the function of GRK5 in pancreatic β cells. We generated a novel pancreatic β cell-specific GRK5 knockout mass (βGRK5 KO) and evaluated glucose tolerance and metabolic changes by body weight measurement, oral glucose tolerance test, and insulin tolerance test. The number of pancreatic β cells was quantified by immunohistochemistry. Glucose loading and Ca2+ imaging was performed on isolated islets to evaluate insulin secretory capacity. To elucidate the mechanism of βGRK5 on β cell mass regulation, we performed RNA-seq of isolated islets and identified the signaling pathways that could be regulated by GRK5. Furthermore, in vitro experiments were conducted using human islets and mouse βGRK5 KO islets to clarify the direct effects of GRK5 on these pathways. βGRK5 KO islets showed impaired glucose tolerance and insulin secretion, but no change in body weight or insulin resistance, suggesting that the main cause of impaired glucose tolerance is impaired insulin secretion. Isolated islets showed no abnormalities in insulin secretory capacity or changes in calcium influx, but histologically showed a decrease in β cell mass. Consistent with the decreased β cell mass in βGRK5 KO, the cell cycle inhibitor gene Cdkn1a was upregulated in βGRK5 KO islets; this phenocopies the βSOX4 KO. Furthermore, we found that Grk5 positively regulates facultative increases in β cell mass through activity-dependent phosphorylation of HDAC5 and subsequent transcription of immediate early genes (IEGs) such as Nr4a1, Fosb, Junb, Arc, Egr1 and Srf. Our results suggest that GRK5 is associated with type 2 diabetes through regulation of β cell mass. GRK5 could become a potential target of cell therapy to preserve functional β cells during the progression towards frank diabetes.

In the progression of diabetes, pancreatic islet β-cells respond to increased metabolic demand with functional compensation, followed by pathogenic decompensation of mitochondria-dependent insulin secretion. It is not clear what mechanisms drive, or control, mitochondrial decompensation. Here, we report that anti-apoptotic Bcl-xL maintains mitochondrial integrity in β-cells under non-apoptotic levels of glucose stress. Prolonged glucose excess causes transcriptional reprogramming of glycolysis and β-cell identity genes, while sensitizing glucose-stimulated Ca2+ signaling and insulin secretion. Deletion of Bcl-xL amplifies this insulin hypersecretion and increases mitochondrial fusion, mitochondrial volume, and oxygen consumption, whereas ATP-coupled respiration and mitochondrial hyperpolarization become impaired. Of note, Bcl-xL-deficient β-cells have impaired Pgc-1α expression, and develop specific defects in the expression of Tfam, mitochondrial ribosomal genes, and OXPHOS components under glucose stress. Bcl-xL limits high glucose-induced mitochondrial ROS (mitoROS) levels and pharmacological normalization of mitoROS in Bcl-xL KO cells rescues glucose-induced defects in mitochondrial gene expression and changes to β-cell identity. Our data identify mitoROS as a primary retrograde driver of transcriptional re-wiring in β-cells exposed to excess glucose, and reveal Bcl-xL as an important safeguard against transcriptional and functional decompensation of β-cell mitochondria. Bcl-xL and mitoROS may thus be viable targets to prevent early β-cell dysfunction and the progression of diabetes.

Pancreatic β-cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialized gene expression program which is established during development and then sustained, with limited flexibility, in terminally differentiated β-cells. Dysregulation of this program is seen in type 2 diabetes (T2D) but mechanisms that preserve gene expression or underlie its dysregulation in mature β-cells are not well resolved. Here we show that trithorax group-dependent histone H3 lysine 4 trimethylation (H3K4me3) maintains expression of genes important for insulin biosynthesis and glucose-responsiveness in β-cells. Transcriptional changes in H3K4me3-deficient β-cells lead to severe hyperglycemia in adult mice. We show that H3K4me3 deficiency leads to a less active and more repressed epigenome profile, which locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We then show that H3K4me3 is re-organized in diabetic Leprdb/db mouse islets in favour of weakly active and disallowed genes at the expense of terminal β-cell markers with broad H3K4me3 peaks. Our results point to key roles of H3K4me3 in maintaining mature β-cell function and establishing a dysfunctional transcriptome in diabetic islets.

Summary Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of actively expressed genes, with genes important for cell identity frequently having exceptionally broad H3K4me3-enriched domains at their TSS. While H3K4 methylation is implicated in contributing to transcription, maintaining transcriptional stability, facilitating enhancer-promoter interactions, and preventing irreversible silencing, some studies suggest it has little functional impact. Therefore, the function of H3K4 methylation is not resolved. Insufficient insulin release by β-cells is the primary etiology in type 2 diabetes (T2D) and is associated with the loss of expression of genes essential to normal β-cell function. We find that H3K4me3 is reduced in islets from mouse models of diabetes and from human donors with T2D. Using a genetic mouse model to impair H3K4 methyltransferase activity of TrxG complexes, we find that reduction of H3K4 methylation significantly reduces insulin production and glucose-responsiveness and increases transcriptional entropy, indicative of a loss of β-cell maturity. Genes that are downregulated by reduction to H3K4 methylation are concordantly downregulated in T2D. Loss of H3K4 methylation causes global dilution of epigenetic complexity but does not generally reduce gene expression – instead, genes related to β-cell function and/or in particular chromatin environments are specifically affected. While neither H3K4me3 nor H3K4me1 are strictly required for the expression of many genes, the expression of genes with critical roles in β-cell function becomes destabilized, with increased variance and decreased overall expression. Our data further suggests that, in absence of H3K4me3, promoter-associated H3K4me1 is sufficient to maintain expression. Together, these data implicate H3K4 methylation dysregulation as destabilizing β-cell gene expression and contributing to β-cell dysfunction in T2D. Competing Interest Statement The authors have declared no competing interest. Footnotes * Meilin An's name has been corrected in the Author list metadata.