Metabolomics Study of the Effects of Inflammation, Hypoxia, and High Glucose on Isolated Human Pancreatic Islets

Garcia‐Contreras, Marta; Tamayo-Garcia, Alejandro; Pappan, Kirk L.; Michelotti, Gregory A.; Stabler, Cherie L.; Ricordi, Camillo; Buchwald, Péter

doi:10.1021/acs.jproteome.7b00160

Cited by 40 publications

(34 citation statements)

References 75 publications

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“…Of these findings, our discovery that KYNA is a more potent agonist of HCAR3 than GPR35 is particularly notable. KYNA is known to play important roles in neuroprotection, depression, schizophrenia, obesity, diabetes, and cancer (13)(14)(15). Prior to this study, the only GPCR known to respond to KYNA was GPR35 (16).…”

Section: New Interactions Between Gpcrs and Amino Acid Metabolitesmentioning

confidence: 99%

DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

Kapolka¹,

Taghon²,

Rowe³

et al. 2020

Preprint

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More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput CRISPR engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan induction by Functional Integrated Receptors: DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCRbarcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and observed new GPCR-ligand interactions with amino acid, lipid, sugar, and steroid metabolites. Remarkably, many of these findings pertained to understudied "pharmacologically dark" receptors GPR4, GPR65, GPR68, and HCAR3. For example, we found that kynurenic acid activated HCAR3 with a nearly 20-fold lower EC50 than GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying novel ligand interactions with prototypic and understudied GPCRs.

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Section: New Interactions Between Gpcrs and Amino Acid Metabolitesmentioning

confidence: 99%

DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

Kapolka¹,

Taghon²,

Rowe³

et al. 2020

Preprint

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“…Lactate, a classic marker of hypoxia, was elevated in islets and culture media after a 24-hour hypoxic condition of 3% O 2 , concomitant with the reduced ability to respond to high glucose. 28 Molecular expression changes in the islets exposed to hypoxic conditions was shown in hypoxia-inducible factor (HIF)–related pathways. 29 – 32 In addition, recent studies suggested the involvement of other mechanisms independent of HIF-1, by showing that the expression of genes associated with β-cell transcription pathways and insulin secretion pathways are altered after exposing isolated mouse islets and mouse β-cell line (MIN6) to hypoxia (<5% O 2 ) for 16 to 30 hours.…”

Section: Effect Of Oxygen On Isolated Isletsmentioning

confidence: 99%

Impact of Oxygen on Pancreatic Islet Survival

Komatsu

Kandeel²,

Mullen³

2018

Pancreas

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Pancreatic islet transplantation is a promising treatment option for individuals with type 1 diabetes; however, maintaining islet function after transplantation remains a large challenge. Multiple factors, including hypoxia associated events, trigger pretransplant and posttransplant loss of islet function. In fact, islets are easily damaged in hypoxic conditions before transplantation including the preparation steps of pancreas procurement, islet isolation, and culture. Furthermore, after transplantation, islets are also exposed to the hypoxic environment of the transplant site until they are vascularized and engrafted. Because islets are exposed to such drastic environmental changes, protective measures are important to maintain islet viability and function. Many studies have demonstrated that the prevention of hypoxia contributes to maintaining islet quality. In this review, we summarize the latest oxygen-related islet physiology, including computational simulation. Furthermore, we review recent advances in oxygen-associated treatment options used as part of the transplant process, including up-to-date oxygen generating biomaterials as well as a classical oxygen inhalation therapy.

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“…Insulin concentrations were determined using commercially available human insulin ELISA kits (Mercodia Inc., Winston Salem, NC, USA) and converted to μg/l using the formula provided by the manufacturer (1 µg/l = 23 mU/l). Because accurate assessment of islet mass (IEQ) is challenging, to account for possible differences among parallel channels, values were adjusted by up to 30% based on the response to KCl as described before using the area under the curve (AUC) in each column for normalization 36 , 37 , 48 , 49 , 56 , 57 . All responses are scaled to 100 IEQ.…”

Section: Methodsmentioning

confidence: 99%

“…Although not part of currently accepted final islet product release criteria, such studies represent the most complex in vitro assay to assess the quality and function of isolated pancreatic islets and provide considerably more physiological data than those obtainable from static GSIS and corresponding stimulation indices (SIs). Improved equipment and analytical techniques now allow the quantitative assessment of insulin release kinetics with customizable temporal resolution and under fully controllable incoming concentrations of glucose and/or other secretagogues of interest 36 , 37 , 44 – 49 . Dynamic perifusion studies are now routinely used to assess the quality and function of islets 46 , 50 , 51 , and microfluidic chip technologies make possible even the quantitative monitoring of single islet insulin secretion with high time resolution 52 , 53 .…”

Section: Introductionmentioning

confidence: 99%

The Effect of Recovery Warm-up Time Following Cold Storage on the Dynamic Glucose-stimulated Insulin Secretion of Isolated Human Islets

et al. 2020

Self Cite

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Standardized islet characterization assays that can provide results in a timely manner are essential for successful islet cell transplantation. A critical component of islet cell quality is β-cell function, and perifusion-based assessments of dynamic glucose-stimulated insulin secretion (GSIS) are the most informative method to assess this, as they provide the most complex in vitro evaluation of GSIS. However, protocols used vary considerably among centers and investigators as they often use different low- and high-glucose concentrations, exposure-times, flow-rates, oxygen concentrations, islet numbers, analytical methods, measurement units, and instruments, which result in different readouts and make comparisons across platforms difficult. Additionally, the conditions of islet storage and shipment prior to assessment may also affect islet function. Establishing improved standardized protocols for perifusion GSIS assays should be an integral part of the ongoing effort to increase the rigor of human islet studies. Here, we performed detailed evaluation of GSIS of human islets using a fully automated multichannel perifusion instrument following various warm-up recovery times after cold storage that corresponds to current shipping conditions (8°C). We found that recovery times shorter than 18 h (overnight) resulted in impaired insulin secretion. While the effects were relatively moderate on second-phase insulin secretion, first-phase peaks were restored only following 18-h incubation. Hence, the biphasic profile of dynamic GSIS was considerably affected when islets were not allowed to recover for a sufficient time after being maintained in cold. Accordingly, while cold storage might improve islet cell survival during shipment and prolong the length of culture, functional assessments should be performed only after allowing for at least overnight recovery at physiological temperatures.

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Metabolomics Study of the Effects of Inflammation, Hypoxia, and High Glucose on Isolated Human Pancreatic Islets

Cited by 40 publications

References 75 publications

DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

Impact of Oxygen on Pancreatic Islet Survival

The Effect of Recovery Warm-up Time Following Cold Storage on the Dynamic Glucose-stimulated Insulin Secretion of Isolated Human Islets

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