CRISPR-based genome editing in primary human pancreatic islet cells

AbstractGene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATPchannel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes inABCC8,SIX2andSIX3expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

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Effects of glucose and d-3-hydroxybutyrate on human pancreatic islet cell function

Clinical Science ◽

10.1042/cs0680567 ◽

1985 ◽

Vol 68 (5) ◽

pp. 567-572 ◽

Cited By ~ 6

Author(s):

C. J. Rhodes ◽

I. L. Campbell ◽

T. M. Szopa ◽

T. J. Biden ◽

P. D. Reynolds ◽

...

Keyword(s):

Insulin Release ◽

Human Tissue ◽

Islet Cell ◽

Cell Function ◽

Human Islets ◽

Β Cell ◽

Pancreatic Islet Cell ◽

Sigmoidal Curve ◽

Β Cell Function ◽

Islet Cell Function

1. β-Cell function in human islets derived from a number of kidney donors was investigated by using various types of islet preparations. 2. With fresh islets, both insulin release and biosynthesis were increased by raising glucose concentrations, although the response was a variable one. 3. In fresh islets, the effects of 5 mmol of glucose/l on release were potentiated by 10 mmol of d-3-hydroxybutyrate/l. 4. Insulin release at 20 mmol of glucose/l was inhibited by adrenaline (0.1 mmol/l), and potentiated by theophylline (10 mmol/l) in the presence of 5 mmol of glucose/l, in islets cultured for 4 days. 5. After culture for 8 days, islets still showed an increase in insulin release and biosynthesis in response to glucose. 6. Pancreas slices derived from fresh human tissue also responded to increasing concentrations of glucose with a sigmoidal curve for insulin release.

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Detailed protocol for evaluation of dynamic perifusion of human islets to assess β-cell function

Islets ◽

10.4161/isl.3.5.15938 ◽

2011 ◽

Vol 3 (5) ◽

pp. 284-290 ◽

Cited By ~ 15

Author(s):

Kwamina Bentsi-Barnes ◽

Máire E. Doyle ◽

Danny Abad ◽

Fouad Kandeel ◽

Ismail Al-Abdullah

Keyword(s):

Cell Function ◽

Human Islets ◽

Β Cell ◽

Β Cell Function ◽

Detailed Protocol

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Resveratrol and curcumin enhance pancreatic β-cell function by inhibiting phosphodiesterase activity

Journal of Endocrinology ◽

10.1530/joe-14-0335 ◽

2014 ◽

Vol 223 (2) ◽

pp. 107-117 ◽

Cited By ~ 53

Author(s):

Michael Rouse ◽

Antoine Younès ◽

Josephine M Egan

Keyword(s):

Insulin Secretion ◽

Cell Lines ◽

Cell Function ◽

Human Islets ◽

Dependent Manner ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Pde Inhibitors ◽

Β Cell Function

Resveratrol (RES) and curcumin (CUR) are polyphenols that are found in fruits and turmeric, and possess medicinal properties that are beneficial in various diseases, such as heart disease, cancer, and type 2 diabetes mellitus (T2DM). Results from recent studies have indicated that their therapeutic properties can be attributed to their anti-inflammatory effects. Owing to reports stating that they protect against β-cell dysfunction, we studied their mechanism(s) of action in β-cells. In T2DM, cAMP plays a critical role in glucose- and incretin-stimulated insulin secretion as well as overall pancreatic β-cell health. A potential therapeutic target in the management of T2DM lies in regulating the activity of phosphodiesterases (PDEs), which degrade cAMP. Both RES and CUR have been reported to act as PDE inhibitors in various cell types, but it remains unknown if they do so in pancreatic β-cells. In our current study, we found that both RES (0.1–10 μmol/l) and CUR (1–100 pmol/l)-regulated insulin secretion under glucose-stimulated conditions. Additionally, treating β-cell lines and human islets with these polyphenols led to increased intracellular cAMP levels in a manner similar to 3-isobutyl-1-methylxanthine, a classic PDE inhibitor. When we investigated the effects of RES and CUR on PDEs, we found that treatment significantly downregulated the mRNA expression of most of the 11 PDE isozymes, including PDE3B, PDE8A, and PDE10A, which have been linked previously to regulation of insulin secretion in islets. Furthermore, RES and CUR inhibited PDE activity in a dose-dependent manner in β-cell lines and human islets. Collectively, we demonstrate a novel role for natural-occurring polyphenols as PDE inhibitors that enhance pancreatic β-cell function.

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Aging-Dependent Demethylation of Regulatory Elements Correlates with Chromatin State and Improved β Cell Function

Cell Metabolism ◽

10.1016/j.cmet.2015.07.025 ◽

2015 ◽

Vol 22 (4) ◽

pp. 619-632 ◽

Cited By ~ 109

Author(s):

Dana Avrahami ◽

Changhong Li ◽

Jia Zhang ◽

Jonathan Schug ◽

Ran Avrahami ◽

...

Keyword(s):

Cell Function ◽

Regulatory Elements ◽

Chromatin State ◽

Β Cell ◽

Β Cell Function

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Intra-islet GLP-1, but not CCK, is necessary for β-cell function in mouse and human islets

Scientific Reports ◽

10.1038/s41598-020-59799-2 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Arnaldo Henrique de Souza ◽

Jiayin Tang ◽

Amanjot Kaur Yadev ◽

Samuel T. Saghafi ◽

Carly R. Kibbe ◽

...

Keyword(s):

Cell Function ◽

Human Islets ◽

Β Cell ◽

Β Cell Function

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Screening enteroviruses for β-cell tropism using foetal porcine β-cells

Journal of General Virology ◽

10.1099/0022-1317-82-8-1909 ◽

2001 ◽

Vol 82 (8) ◽

pp. 1909-1916 ◽

Cited By ~ 7

Author(s):

Merja Roivainen ◽

Petri Ylipaasto ◽

Jarkko Ustinov ◽

Tapani Hovi ◽

Timo Otonkoski

Keyword(s):

Cell Function ◽

Biological Properties ◽

Human Islets ◽

Cell Tropism ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Systematic Screening ◽

Adult Human ◽

Β Cell Function

Primary adult human insulin-producing β-cells are susceptible to infection by prototype strains of coxsackieviruses (CV) and infection may result in impaired β-cell function and/or cell death, as shown for coxsackie B virus (CVB) types 4 and 5, or have no apparent immediate adverse effects, as shown for CVA-9. Because of the limited availability of human pancreatic β-cells, the aim of this study was to find out if foetal porcine pancreatic islets could be used as a substitute in enterovirus (EV) screening. These cells resemble human β-cells in several biological properties. CVB infection resulted in a rapid progressive decline of insulin content and reponsiveness to insulin release. The amount of virus inoculum sufficient for this destruction was small, corresponding to only 55 infectious units per pancreas. In contrast to CVBs, CVA-9 replicated poorly, and sometimes not at all, in foetal porcine β-cells. The first signs of functional impairment and cell destruction, if present at all, were seen only after 1–3 weeks of incubation. Furthermore, CVA-16, several strains of echoviruses and human parechovirus type 1 were unable to replicate in foetal porcine pancreatic β-cells. Based on these results, foetal porcine islets are somewhat more sensitive to CVB infection than adult human islets, whereas many other human EV strains do not infect porcine β-cells. Therefore, foetal porcine β-cells cannot be used for systematic screening of human EV strains and isolates for β-cell tropism, but they might provide a useful model for detailed studies on the interaction of CVBs with β-cells.

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Copine 3 “CPNE3” is a novel regulator for insulin secretion and glucose uptake in pancreatic β-cells

Scientific Reports ◽

10.1038/s41598-021-00255-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Waseem El-Huneidi ◽

Shabana Anjum ◽

Abdul Khader Mohammed ◽

Hema Unnikannan ◽

Rania Saeed ◽

...

Keyword(s):

Insulin Secretion ◽

Glucose Uptake ◽

Cancer Progression ◽

Cell Function ◽

Human Islets ◽

Glucose Sensing ◽

Β Cell ◽

Β Cell Function ◽

Novel Target

AbstractCopine 3 (CPNE3) is a calcium-dependent phospholipid-binding protein that has been found to play an essential role in cancer progression and stages. However, its role in pancreatic β-cell function has not been investigated. Therefore, we performed a serial of bioinformatics and functional experiments to explore the potential role of Cpne3 on insulin secretion and β-cell function in human islets and INS-1 (832/13) cells. RNA sequencing and microarray data revealed that CPNE3 is highly expressed in human islets compared to other CPNE genes. In addition, expression of CPNE3 was inversely correlated with HbA1c and reduced in human islets from hyperglycemic donors. Silencing of Cpne3 in INS-1 cells impaired glucose-stimulated insulin secretion (GSIS), insulin content and glucose uptake efficiency without affecting cell viability or inducing apoptosis. Moreover, mRNA and protein expression of the key regulators in glucose sensing and insulin secretion (Insulin, GLUT2, NeuroD1, and INSR) were downregulated in Cpne3-silenced cells. Taken together, data from the present study provides a new understanding of the role of CPNE3 in maintaining normal β-cell function, which might contribute to developing a novel target for future management of type 2 diabetes therapy.

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