Biology subjects

Pasula, D. J.

Publications and source records attributed to Pasula, D. J..

4 recordsLinked to original sources

bioRxiv2026

Autophagy protects pancreatic β-cells during hypoxia and islet transplantation but is compromised by TFEB-lysosomal dysfunction

Hypoxia is a potent stressor and a major cause of {beta}-cell failure and loss after islet transplantation. Autophagy is a critical homeostatic mechanism that preserves organelle integrity and metabolic balance in cells under stress, but whether it supports {beta}-cell adaptation to sustained oxygen deprivation is unclear. Here, we used {beta}-cell-specific Atg5 knockout together with hypoxia and transplantation models, to demonstrate that autophagy is a major determinant of {beta}-cell survival during oxygen limitation and supports islet graft function. However, prolonged hypoxia suppressed autophagic flux, reduced lysosomal activity, and led to autophagosome accumulation, indicating failure of the lysosomal clearance pathway. This was accompanied by a marked reduction in transcription factor EB (TFEB) and its lysosomal target genes. Genetic and pharmacological activation of TFEB restored lysosomal gene expression and cathepsin B activity and improved {beta}-cell viability under hypoxia, implicating TFEB decline as a contributor to autophagy-lysosome dysfunction. Together, these findings outline a sequence in which autophagy initially safeguards {beta}-cells but becomes ineffective under sustained hypoxia as TFEB levels fall, identifying TFEB as a potential target to strengthen {beta}-cell resilience and survival in islet transplantation.

cell biology↗

bioRxiv2022

Type 2 diabetes susceptibility gene GRK5 regulates physiological pancreatic β-cell proliferation via phosphorylation of HDAC5 in mice

ObjectiveDiabetes onset is accompanied with {beta}-cell deficiency, and thus restoring functional {beta}-cell mass is a promising therapy for those with diabetes. However, the regulatory mechanisms controlling {beta}-cell mass are not fully understood. Previously, we demonstrated that the transcription factor SOX4 is required for {beta}-cell proliferation in the prediabetic state. To elucidate potential mechanisms by which SOX4 regulates {beta}-cell mass, we performed RNA sequencing (RNA-seq) using pancreatic {beta}-cell specific SOX4 knockout mice ({beta}SOX4 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 {beta} cells. MethodsWe generated a novel pancreatic {beta} cell-specific GRK5 knockout mass ({beta}GRK5 KO) and evaluated glucose tolerance and metabolic changes by body weight measurement, oral glucose tolerance test, and insulin tolerance test. The number of pancreatic {beta} 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 {beta}GRK5 on {beta} 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 {beta}GRK5 KO islets to clarify the direct effects of GRK5 on these pathways. Results{beta}GRK5 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 {beta} cell mass. Consistent with the decreased {beta} cell mass in {beta}GRK5 KO, the cell cycle inhibitor gene Cdkn1a was upregulated in {beta}GRK5 KO islets; this phenocopies the {beta}SOX4 KO. Furthermore, we found that Grk5 positively regulates facultative increases in {beta} 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. ConclusionsOur results suggest that GRK5 is associated with type 2 diabetes through regulation of {beta} cell mass. GRK5 could become a potential target of cell therapy to preserve functional {beta} cells during the progression towards frank diabetes.

physiology↗

bioRxiv2021

Bcl-xL restricts transcriptional, morphological and functional decompensation of β-cell mitochondria under chronic glucose excess

In the progression of diabetes, pancreatic islet {beta}-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 {beta}-cells under non-apoptotic levels of glucose stress. Prolonged glucose excess causes transcriptional reprogramming of glycolysis and {beta}-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 {beta}-cells have impaired Pgc-1 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 {beta}-cell identity. Our data identify mitoROS as a primary retrograde driver of transcriptional re-wiring in {beta}-cells exposed to excess glucose, and reveal Bcl-xL as an important safeguard against transcriptional and functional decompensation of {beta}-cell mitochondria. Bcl-xL and mitoROS may thus be viable targets to prevent early {beta}-cell dysfunction and the progression of diabetes.

cell biology↗

bioRxiv2021

H3K4 Methylation in β-cells Prevents Transcriptional Downregulation and Variance Associated with Type 2 Diabetes

Pancreatic {beta}-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 {beta}-cells. Dysregulation of this program is seen in type 2 diabetes (T2D) but mechanisms that preserve gene expression or underlie its dysregulation in mature {beta}-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 {beta}-cells. Transcriptional changes in H3K4me3-deficient {beta}-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 {beta}-cell markers with broad H3K4me3 peaks. Our results point to key roles of H3K4me3 in maintaining mature {beta}-cell function and establishing a dysfunctional transcriptome in diabetic islets.

cell biology↗