Clinical Islet Isolation

Hawthorne, Wayne J.; Williams, Lindy; Chew, Yi Vee

doi:10.1007/978-3-319-39824-2_7

Cited by 7 publications

(4 citation statements)

References 182 publications

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“…Human islets isolated as described [27] were suspended in RPMI1640 media, 11 mM glucose, 10% fetal bovine serum for overnight incubation then dispersed in TrypLE Express for 3 min, briefly dissociated into single cells by pipetting, resuspended into 5 volumes of supplemented RPMI1640, then placed on glass coverslips. After overnight incubation, cells were washed twice in Wash Buffer (0.1% BSA in PBS with 0.01% sodium azide), fixed at ambient temperature for 20 min with 4% paraformaldehyde in PBS, then washed twice in Wash Buffer before antigen retrieval in 0.1% SDS for 5 min.…”

Section: Human Islet Recovery Dispersion and Immunocytochemistrymentioning

confidence: 99%

Fibroblast activation protein enzyme deficiency prevents liver steatosis, insulin resistance and glucose intolerance and increases fibroblast growth factor-21 in diet induced obese mice

Chowdhury

Song

Zhang

et al. 2018

Preprint

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Background & AimsFibroblast activation protein-a (FAP) is a post-proline peptidase closely related to dipeptidyl peptidase-4. FAP degrades bioactive peptides including fibroblast growth factor-21 (FGF-21) and neuropeptide Y. We examined metabolic outcomes of specific genetic ablation of FAP and its enzyme activity in a mouse model of diet-induced obesity (DIO) causing fatty liver.MethodsWildtype (WT) and genetically modified FAP deficient mice that specifically lacked either the FAP protein or FAP enzyme activity received chow, or an atherogenic diet for 8 to 20 weeks of DIO.ResultsFAP deficient male and female mice in the DIO model were more metabolically healthy than controls. The FAP deficient mice had less glucose intolerance, liver lipid, adiposity, insulin resistance, pancreatic and plasma insulin, pancreatic β-cell hyperplasia, serum alanine transaminase and circulating cholesterol compared to wild type controls. Furthermore, FAP deficiency lowered respiratory exchange ratio and greatly increased intrahepatic non-esterified free fatty acids, indicative of increased lipolysis and β-oxidation. Concordantly, lipogenic genes (Pparg, Gck, Acc, Fasn) and hepatic triglyceride and fatty acid uptake genes (Cd36, Apoc3, Ldlr) and plasma low-density lipoprotein cholesterol were downregulated. Glucagon like peptide-1 levels were unaltered. FAP was localized to human pancreatic β-cells and pancreas from diabetes mellitus patients contained elevated FAP activity. Comparable data from a FAP gene knockout mouse and a novel mouse lacking FAP enzyme activity indicated that these metabolic changes depended upon the enzymatic activity of FAP. These changes may be driven by FGF-21, which was upregulated in livers of FAP deficient DIO mice.ConclusionThis is the first study to show that specific genetic ablation of FAP activity or protein protects against DIO-driven glucose intolerance, hyperinsulinaemia, insulin resistance, hypercholesterolaemia and liver steatosis in mice and provide mechanistic insights.

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Section: Human Islet Recovery Dispersion and Immunocytochemistrymentioning

confidence: 99%

Fibroblast activation protein enzyme deficiency prevents liver steatosis, insulin resistance and glucose intolerance and increases fibroblast growth factor-21 in diet induced obese mice

Chowdhury

Song

Zhang

et al. 2018

Preprint

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“…21,22 The isolation procedure has been refined and optimized over time to obtain highly functional, pure islet preparations for clinical transplantation. 23 The success of the clinical transplantation relies on minimal damage to the exocrine pancreas during pancreas preservation and digestion to minimize the release of endogenous proteases from acinar cells. It also relies on optimal separation of islets from acinar cells to prevent protease damage to islets and to improve viability and function of isolated islet cells.…”

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confidence: 99%

Human Pancreatic Acinar Cells

Lugea

Waldron

Mareninova

et al. 2017

The American Journal of Pathology

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Knowledge of the molecular mechanisms of acute pancreatitis is largely based on studies using rodents. To assess similar mechanisms in humans, we performed ex vivo pancreatitis studies in human acini isolated from cadaveric pancreata from organ donors. Because data on these human acinar preparations are sparse, we assessed their functional integrity and cellular and organellar morphology using light, fluorescence, and electron microscopy; and their proteome by liquid chromatography-tandem mass spectrometry. Acinar cell responses to the muscarinic agonist carbachol (CCh) and the bile acid taurolithocholic acid 3-sulfate were also analyzed. Proteomic analysis of acini from donors of diverse ethnicity showed similar profiles of digestive enzymes and proteins involved in translation, secretion, and endolysosomal function. Human acini preferentially expressed the muscarinic acetylcholine receptor M3 and maintained physiological responses to CCh for at least 20 hours. As in rodent acini, human acini exposed to toxic concentrations of CCh and taurolithocholic acid 3-sulfate responded with trypsinogen activation, decreased cell viability, organelle damage manifest by mitochondrial depolarization, disordered autophagy, and pathological endoplasmic reticulum stress. Human acini also secreted inflammatory mediators elevated in acute pancreatitis patients, including IL-6, tumor necrosis factor-α, IL-1β, chemokine (C-C motif) ligands 2 and 3, macrophage inhibitory factor, and chemokines mediating neutrophil and monocyte infiltration. In conclusion, human cadaveric pancreatic acini maintain physiological functions and have similar pathological responses and organellar disorders with pancreatitis-causing treatments as observed in rodent acini.

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“…Three litters of mice were used containing 7 (2 controls, 5 mutant), 6 (2 controls, 4 mutant) and 8 ( Human pancreata were obtained, with informed consent from next of kin, from heartbeating, brain-dead donors. Human islets were purified by intraductal perfusion and digestion of the pancreas with collagenase followed by purification using Ficoll density gradients [47]. Purified islets were cultured in Connaught Medical Research Laboratories (CMRL) 1066 medium (Invitrogen) supplemented with 4% human serum albumin, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine (complete CMRL), in a 37 °C, 5 % CO2 humidified incubator.…”

Section: Micementioning

confidence: 99%

A fluorescent timer reporter enables sorting of insulin secretory granules by age

Yau

Hays

Liang

et al. 2020

Journal of Biological Chemistry

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Within the pancreatic β-cells, insulin secretory granules (SGs) exist in functionally distinct pools, displaying variations in motility as well as docking and fusion capability. Current therapies that increase insulin secretion do not consider the existence of these distinct SG pools. Accordingly, these approaches are effective only for a short period, with a worsening of glycemia associated with continued decline in β-cell function. Insulin granule age is underappreciated as a determinant for why an insulin granule is selected for secretion and may explain why newly synthesized insulin is preferentially secreted from β-cells. Here, using a novel fluorescent timer protein, we aimed to investigate the preferential secretion model of insulin secretion and identify how granule aging is affected by variation in the β-cell environment, such as hyperglycemia. We demonstrate the use of a fluorescent timer construct, syncollin-dsRedE5TIMER, which changes its fluorescence from green to red over 18 h, in both microscopy and fluorescence-assisted organelle-sorting techniques. We confirm that the SG-targeting construct localizes to insulin granules in β-cells and does not interfere with normal insulin SG behavior. We visualize insulin SG aging behavior in MIN6 and INS1 β-cell lines and in primary C57BL/6J mouse and nondiabetic human islet cells. Finally, we separated young and old insulin SGs, revealing that preferential secretion of younger granules occurs in glucose-stimulated insulin secretion. We also show that SG population age is modulated by the β-cell environment in vivo in the db/db mouse islets and ex vivo in C57BL/6J islets exposed to different glucose environments.

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Clinical Islet Isolation

Cited by 7 publications

References 182 publications

Fibroblast activation protein enzyme deficiency prevents liver steatosis, insulin resistance and glucose intolerance and increases fibroblast growth factor-21 in diet induced obese mice

Fibroblast activation protein enzyme deficiency prevents liver steatosis, insulin resistance and glucose intolerance and increases fibroblast growth factor-21 in diet induced obese mice

Human Pancreatic Acinar Cells

A fluorescent timer reporter enables sorting of insulin secretory granules by age

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