Biology subjects

Bone, R. N.

Publications and source records attributed to Bone, R. N..

4 recordsLinked to original sources

bioRxiv2022

SERCA2 regulates proinsulin processing and processing enzyme maturation in the pancreatic β cell

Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in both type 1 and type 2 diabetes; however, the mechanisms underlying impaired proinsulin processing remain incompletely understood. Here, we identify the sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) pump and {beta} cell ER Ca2+ as key regulators of systemic glucose tolerance and proinsulin processing. We generated mice with a {beta} cell-specific SERCA2 deletion ({beta}S2KO) and SERCA2 deficient INS-1 cells to show that SERCA2 loss increases systemic and pancreatic levels of proinsulin protein and leads to aberrant localization of proinsulin within the proximal {beta} cell secretory pathway. These defects in proinsulin processing were linked to reduced maturation of the proinsulin processing enzymes PC1/3 and PC2, suggesting a model whereby chronic ER Ca2+ depletion in the {beta} cell, which is observed in many pathological conditions, impairs the spatial regulation of prohormone trafficking, processing, and maturation within the {beta} cell secretory pathway.

physiology↗

bioRxiv2021

A discovery-based proteomics approach identifies protein disulfide isomerase (PDIA1) as a biomarker of β cell stress in type 1 diabetes

BackgroundActivation of stress pathways intrinsic to the {beta} cell are thought to both accelerate {beta} cell death and increase {beta} cell immunogenicity in type 1 diabetes (T1D). However, information on the timing and scope of these responses is lacking. MethodsTo identify temporal and disease-related changes in islet {beta} cell protein expression, data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer. FindingsIn islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in the maintenance of {beta} cell function and stress mitigation, followed by loss of expression of protective proteins that heralded diabetes onset. Pathway analysis identified EIF2 signaling and the unfolded protein response, mTOR signaling, mitochondrial function, and oxidative phosphorylation as commonly modulated pathways in both diabetic NOD mice and NOD-SCID mice rendered acutely diabetic by adoptive transfer, highlighting this core set of pathways in T1D pathogenesis. In immunofluorescence validation studies, {beta} cell expression of protein disulfide isomerase A1 (PDIA1) and 14-3-3b were found to be increased during disease progression in NOD islets, while PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to age and sex-matched non-diabetic controls. InterpretationWe identified a common and core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of {beta} cell stress in T1D.

physiology↗

bioRxiv2021

Loss of Secretory Pathway Ca2+ ATPase (SPCA1) Impairs Insulin Secretion and Reduces Autophagy in the Pancreatic Islet

The {beta} cell Golgi apparatus serves as a significant store of intracellular Ca2+ and an important site of proinsulin maturation. However, the contribution of Golgi Ca2+ to diabetes pathophysiology is unknown. The Golgi primarily utilizes the Secretory Pathway Ca2+ ATPase (SPCA1) to maintain intraluminal Ca2+ stores, and loss of SPCA1 has been linked to impaired Golgi function in other cell types. Here, we demonstrated that SPCA1 expression is decreased in islets from diabetic mice and human organ donors with type 2 diabetes, suggesting SPCA1 may impact diabetes development. INS-1 {beta} cells lacking SPCA1 (SPCA1KO) showed reduced intraluminal Golgi Ca2+ levels, reduced glucose-stimulated insulin secretion (GSIS), and increased insulin content. Islets from SPCA1 haploinsufficient mice (SPCA1+/-) exhibited reduced GSIS, altered glucose-induced Ca2+ oscillations, and altered insulin granule maturation. Autophagy can regulate granule homeostasis, therefore we induced autophagy with Torin1 and found that SPCA1KO cells and SPCA1+/- islets had reduced levels of the autophagosome marker LC3-II. Furthermore, SPCA1KO LC3-II were unchanged after blocking autophagy initiation or autophagolysosome fusion and acidification. Thus, we concluded that {beta} cell SPCA1 plays an important role in the maintenance of Golgi Ca2+ homeostasis and reduced Golgi Ca2+ impairs autophagy initiation and may impact insulin granule homeostasis.

cell biology↗

bioRxiv2019

Cigarette Smoke Exposure Impairs β-Cell Function Through Activation of Oxidative Stress and Ceramide Accumulation

ObjectivesEpidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic {beta}-cell function using rodent and in vitro models.\n\nMethodsBeginning at 8 weeks of age, C57BL/6J mice were concurrently fed high fat-diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD exposure. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured {beta}-cells (INS-1) and primary islets were exposed ex vivo to CS extract (CSE), and {beta}-cell function and viability were tested. Since CS increases ceramide in lungs cells and these bioactive sphingolipids have been implicated in pancreatic {beta}-cell dysfunction in diabetes, islet and {beta}-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry.\n\nResultsCompared to HFD-fed ambient air-exposed mice, HFD-fed and CS- exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and a significantly greater increase in glucose intolerance compared to non-smoking control mice. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of {beta}-cell dysfunction, significantly lower {beta}-cell mass (p=0.02), and reduced {beta}-cell proliferation (p=0.006), and increased islet ceramide accumulation. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide accumulation, reduce insulin gene expression and glucose-stimulated insulin secretion, and increase {beta}-cell oxidative and ER stress. Treatment with the antioxidant N-acetylcysteine, markedly attenuated the effects of CSE on ceramide levels, restored {beta}-cell function and survival, and increased cyclin D2 expression, while also reducing activation of {beta}-cell ER and oxidative stress.\n\nConclusionsOur results indicate that CS exposure inhibits insulin production, processing, and secretion and reduced {beta}-cell viability and proliferation. These effects were linked to increased {beta}-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of mechanisms by which CS exposure impairs {beta}-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.

molecular biology↗