β-cell-specific deletion of PFKFB3 restores cell fitness competition and physiological replication under diabetogenic stress

HIF1α and PFKFB3 play a critical role in the survival of damaged β-cells in type–2 diabetes while rendering β-cells non-responsive to glucose stimulation. To discriminate the role of PFKFB3 from HIF1α in vivo, we generated mice with conditional β-cell specific disruption of the Pfkfb3 gene on a human islet pancreatic polypeptide (hIAPP+/−) background and a highfat diet (HFD) [PFKFB3βKO + diabetogenic stress (DS)]. PFKFB3 disruption in β-cells under DS led to selective purging of hIAPP-damaged β-cells and the disappearance of insulin- and glucagon positive bihormonal cells. PFKFB3 disruption induced a three-fold increase in β-cell replication as evidenced by minichromosome maintenance 2 protein (MCM2) expression. Unlike high-, lower DS or switch to restricted chow diet abolished HIF1α levels and reversed glucose intolerance of PFKFB3βKO DS mice. Our data suggest that replication and functional recovery of β-cells under DS depend on β-cell competitive and selective purification of HIF1α and PFKFB3-positive β-cells.Larry Hillblom Foundationhttps://www.nature.com/commsbiodm2022Pharmacolog

p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

<p>DNA replication initiation is a key event in the cell cycle, which is dependent on 2 kinases - CDK2 and CDC7. Here we report a novel mechanism in which p53 induces G1 checkpoint and cell cycle arrest by downregulating CDC7 kinase in response to genotoxic stress. We demonstrate that p53 controls CDC7 stability post-transcriptionally via miR-192/215 and post-translationally via Fbxw7β E3 ubiquitin ligase. The p53-dependent pathway of CDC7 downregulation is interlinked with the p53-p21-CDK2 pathway, as p21-mediated inhibition of CDK2-dependent phosphorylation of CDC7 on Thr376 is required for GSK3ß-phosphorylation and Fbxw7ß-dependent degradation of CDC7. Notably, sustained oncogenic high levels of active CDC7 exert a negative feedback onto p53, leading to unrestrained S-phase progression and accumulation of DNA damage. Thus, p53-dependent control of CDC7 levels is essential for blocking G1/S cell-cycle transition upon genotoxic stress, thereby safeguarding the genome from instability and thus representing a novel general stress response.</p

Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations

Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations

Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations

Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations

Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations

Activation of the HIF1α/PFKFB3 stress response pathway in beta cells in type 1 diabetes.

Aims/hypothesisThe conserved hypoxia inducible factor 1 α (HIF1α) injury-response pro-survival pathway has recently been implicated in early beta cell dysfunction but slow beta cell loss in type 2 diabetes. We hypothesised that the unexplained prolonged prediabetes phase in type 1 diabetes may also be, in part, due to activation of the HIF1α signalling pathway.MethodsRNA sequencing (RNA-Seq) data from human islets with type 1 diabetes or after cytokine exposure in vitro was evaluated for activation of HIF1α targets. This was corroborated by immunostaining human pancreases from individuals with type 1 diabetes for 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), the key effector of HIF1α-mediated metabolic remodelling, and by western blotting of islets and INS-1 832/13 cells exposed to cytokines implicated in type 1 diabetes.ResultsHIF1α signalling is activated (p = 4.5 × 10-9) in islets from individuals with type 1 diabetes, and in human islets exposed in vitro to cytokines implicated in type 1 diabetes (p = 1.1 × 10-14). Expression of PFKFB3 is increased fivefold (p &lt; 0.01) in beta cells in type 1 diabetes and in human and rat islets exposed to cytokines that induced increased lactate production. HIF1α attenuates cytokine-induced cell death in beta cells.Conclusions/interpretationThe conserved pro-survival HIF1α-mediated injury-response signalling is activated in beta cells in type 1 diabetes and likely contributes to the relatively slow rate of beta cell loss at the expense of early defective glucose-induced insulin secretion

Cell cycle-related metabolism and mitochondrial dynamics in a replication-competent pancreatic beta-cell line.

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle