Biology subjects

Luciani, D. S.

Publications and source records attributed to Luciani, D. S..

7 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↗

bioRxiv2020

Beta-cell specific insulin resistance promotes glucose-stimulated insulin hypersecretion

Abstract Insulin receptor (Insr) protein can be found at higher levels in pancreatic {beta}-cells than in most other tissues, but the consequences of {beta}-cell insulin resistance remain enigmatic. Ins1cre allele was used to delete Insr specifically in {beta}-cells of both female and male mice. Experimental mice were compared to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined {beta}-cells revealed transcriptomic consequences of Insr loss, which differed between female and male mice. Action potential and calcium oscillation frequencies were increased in Insr knockout {beta}- cells from female, but not male mice, whereas only male {beta}InsrKO mice had reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female {beta}InsrKO and {beta}InsrHET mice exhibited elevated insulin release in perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr did not alter {beta}-cell area up to 9 months of age, nor did it impair hyperglycemia-induced proliferation. Based on our data, we adapted a mathematical model to include {beta}-cell insulin resistance, which predicted that {beta}-cell Insr knockout would improve glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance was significantly improved in female {beta}InsrKO and {beta}InsrHET mice when compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We did not observe improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of {beta}-cell specific insulin resistance. The propensity for hyperinsulinemia was associated with mildly reduced fasting glucose and increased body weight. We further validated our main in vivo findings using the Ins1-CreERT transgenic line and found that male mice had improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that loss of {beta}-cell Insr contributes to glucose-induced hyperinsulinemia, thereby improving glucose homeostasis in otherwise insulin sensitive dietary and age contexts.

physiology↗

bioRxiv2019

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress

ER stress and apoptosis contribute to the loss of pancreatic {beta}-cells under the pro-diabetic conditions of glucolipotoxicity. Although activation of the canonical pathway of intrinsic apoptosis is known to require Bax and Bak, their individual and combined involvement in glucolipotoxic {beta}-cell death have not been demonstrated. It has also remained an open question if Bax and Bak in {beta}-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined {beta}-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic {beta}-cell death in vitro happens in sequential stages; first via non-apoptotic mechanisms and later by apoptosis, which Bax and Bak were redundant in triggering. In contrast, they had non-redundant roles in mediating staurosporine-induced {beta}-cell apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated {beta}-cell Ca2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that Bax and Bak together dampen the unfolded protein response in {beta}-cells during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings identify novel roles of the canonical apoptosis machinery in modulating stress signals that are important for the pathobiology of {beta}-cells in diabetes.

cell biology↗

bioRxiv2019

A pipeline for multidimensional confocal analysis of mitochondrial morphology, function and dynamics in pancreatic β-cells

Live-cell imaging of mitochondrial function and dynamics can provide vital insights into both physiology and pathophysiology, including of metabolic diseases like type 2 diabetes. However, without super-resolution microscopy and commercial analysis software it is challenging to accurately extract features from dense multi-layered mitochondrial networks, such as those in insulin-secreting pancreatic {beta}-cells. Motivated by this, we developed a comprehensive pipeline, and associated ImageJ plugin, that enables 2D/3D quantification of mitochondrial network morphology and dynamics in mouse {beta}-cells, and by extension other similarly challenging cell-types. The approach is based on standard confocal microscopy and shareware, making it widely accessible. The pipeline was validated using mitochondrial photo-labelling and unsupervised cluster analysis, and is capable of morphological and functional analyses on a per-organelle basis, including in 4D (xyzt). Overall, this tool offers a powerful framework for multiplexed analysis of mitochondrial state/function, and provides a valuable resource to accelerate mitochondrial research in health and disease.

cell biology↗