Ismail H. Al-Abdullah

Insulin gene expression is regulated by DNA methylation.

Background Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. Methodology/Principal Findings Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. Conclusions/Significance Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Glucose‐Stimulated Increment in Oxygen Consumption Rate as a Standardized Test of Human Islet Quality

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (ΔOCRglc) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ΔOCRglc obtained at both institutions were compared. Islet specificity was determined by comparing ΔOCRglc in islet and nonislet tissue. The ΔOCRglc ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (ΔOCRglc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ΔOCRglc was statistically independent of percent beta cells, demonstrating that ΔOCRglc is governed more by islet quality than by islet composition. The ΔOCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ΔOCRglc as a measurement of islet quality for human islet transplantation.

PTEN CONTROLS β-CELL REGENERATION IN AGED MICE BY REGULATING CELL CYCLE INHIBITOR P16INK4A

Tissue regeneration diminishes with age, concurrent with declining hormone levels including growth factors such as insulin‐like growth factor‐1 (IGF‐1). We investigated the molecular basis for such decline in pancreatic β‐cells where loss of proliferation occurs early in age and is proposed to contribute to the pathogenesis of diabetes. We studied the regeneration capacity of β‐cells in mouse model where PI3K/AKT pathway downstream of insulin/IGF‐1 signaling is upregulated by genetic deletion of Pten (phosphatase and tensin homologue deleted on chromosome 10) specifically in insulin‐producing cells. In this model, PTEN loss prevents the decline in proliferation capacity in aged β‐cells and restores the ability of aged β‐cells to respond to injury‐induced regeneration. Using several animal and cell models where we can manipulate PTEN expression, we found that PTEN blocks cell cycle re‐entry through a novel pathway leading to an increase in p16ink4a, a cell cycle inhibitor characterized for its role in cellular senescence/aging. A downregulation in p16ink4a occurs when PTEN is lost as a result of cyclin D1 induction and the activation of E2F transcription factors. The activation of E2F transcriptional factors leads to methylation of p16ink4a promoter, an event that is mediated by the upregulation of polycomb protein, Ezh2. These analyses establish a novel PTEN/cyclin D1/E2F/Ezh2/p16ink4a signaling network responsible for the aging process and provide specific evidence for a molecular paradigm that explain how decline in growth factor signals such as IGF‐1 (through PTEN/PI3K signaling) may control regeneration and the lack thereof in aging cells.

Quantitative assessment of β-cell apoptosis and cell composition of isolated, undisrupted human islets by laser scanning cytometry.

Background. Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent &bgr;-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing &bgr;-cell apoptosis and islet cell composition on serial sections of intact isolated islets. Methods. Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Results. Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic &bgr; cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic &bgr; cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. Conclusions. The analysis of whole, nondissociated islets for cell composition and &bgr;-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.

Improvement of human islet cryopreservation by a p38 MAPK inhibitor.

The activation of p38 mitogen‐activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum‐free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD‐282 (ICS‐p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS‐p38IH and islet viability was not significantly different in the three groups. β Cell numbers and function were the highest in islets cryopreserved with ICS‐p38IH. Glucose‐stimulated human C‐peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

The Effects of Digestion Enzymes on Islet Viability and Cellular Composition

The choice of enzyme blend is critical for successful islet isolation. Islet yield, viability, integrity, and function are important factors that influence the outcome of islet transplantation. Liberase HI has been used as a standard enzyme for pancreas digestion and has successfully produced islets that reversed diabetes. However, the replacement of Liberase HI with collagenase NB1 has significantly influenced the process outcome, both in quality and quantity of the isolated islets. The assessment of islet cells by Flow Cytometry (FC) has been reported to be useful for evaluating islet quality. The aim of this study was to assess the isolation outcomes and islet quality when comparing human islet cell processed with Liberase HI and NB1. A total of 66 islet isolations, 46 processed using Liberase HI and 20 using Serva NB1, were retrospectively analyzed. Islet yield, function in vitro, islet cell viability by FC, as well as isolation-related factors were compared. There was no significant difference in donor characteristics such as age and height; however, body mass index (BMI) in the Liberase HI group was significantly higher. There was also no significant difference in prepurification, postisolation, or postculture IEQ or percent recovery between the two groups. Flow data showed Liberase HI preparations had a significantly higher percent of live cells (DAPI-) and NG+/ TMRE+ when compared to NB1. Stimulation Indices (SI) for Liberase HI (n = 45) showed 3.17 and NB1 (n = 18) 2.71 (p = NS). The results of Annexin V/DAPI staining for live, apoptotic, and necrotic cells were 50.7 ± 2.24%, 14.4 ± 1.02%, and 27.8 ± 1.92% for Liberase HI versus 48.1 ± 1.93%, 12.3 ± 0.92%, and 33.9 ± 2.28% for NB1. Islets isolated using Liberase HI showed higher viable β cells by NG/TMRE staining and decreased necrosis by Annexin V/DAPI staining. FC assessment may be useful for determining the choice of digestion enzyme to maximize viable islets.

The role of microRNAs in islet β-cell development

Cell‐based therapies suggest novel treatments to overcome the complication of the current therapeutic approaches in diabetes mellitus type 1. Replacement of the destroyed pancreatic islet β‐cells by appropriate alternative cells needs an efficient approach to differentiate the cells into viable and functional insulin producing cells. Small non‐coding RNA molecules, microRNAs (miRNA), have critical roles in post‐transcriptional regulation of gene expression. Therefore, they can direct the cells toward β‐cell like cells and control islet β‐cell development. Previous reports showed the manipulation of the miRNA expression on islet β‐cell differentiation and regeneration. Likewise, the regulation of epithelial to mesenchymal transition by the miR‐30 family and the miR‐200 family may be a useful approach to conduct islet β‐cell development. Investigation of stem cells differentiation showed that the dynamic expression patterns of miR‐375 and miR‐7 are similar to developing human fetal pancreas while dynamic expression of miR‐146a and miR‐34a occurred during the differentiation. Moreover, miR‐342 and its both targets, FOXA2 and MAFB, are found in β‐cell differentiation and maturation. Because miRNAs can target specific transcription factors during islet β‐cell development and differentiation, they could be offerred as alternative regenerative treatment for diabetes mellitus. Considering that the application of these non‐coding RNAs remains limited in the literature, in this review article, we present an overview of the roles of miRNAs in the islet β‐cell development, focusing on the application of different miRNAs in the experimental protocols.

The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

Background We evaluated 3 commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality after isolation. Methods Retrospectively compared and analyzed islet isolations from pancreata using 3 different enzyme groups: liberase HI (n = 63), collagenase NB1/neutral protease (NP) (n = 43), and liberase mammalian tissue-free collagenase/thermolysin (MTF C/T) (n = 115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate, and in vivo transplantation model in mice. Results Donor characteristics were not significantly different among the 3 enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index, hemoglobin A1c, cold ischemia time, and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the liberase MTF C/T group (73.5 ± 1.5 %) when compared to the liberase HI group (63.6 ± 2.3 %) (P < 0.001) and the collagenase NB1/NP group (61.7 ± 2.9%) (P < 0.001). The stimulation index for GSIS was significantly higher in the liberase MTF C/T group (5.3 ± 0.5) as compared to the liberase HI (2.9 ± 0.2) (P < 0.0001) and the collagenase NB1/NP (3.6 ± 2.9) (P = 0.012) groups. Furthermore, the liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic non-obese diabetic severe combined immunodeficiency mice (65%), which was significantly higher than the liberase HI (42%, P = 0.001) and the collagenase NB1/NP enzymes (41%, P < 0.001). Conclusions Liberase MTF C/T is superior to liberase HI and collagenase NB1/NP in terms of digestion efficacy and GSIS in vitro. Moreover, liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to liberase HI and collagenase NB1/NP enzymes.

Human Pancreatic Islets Isolated From Donors With Elevated HbA1c Levels: Islet Yield and Graft Efficacy.

The aim of this study was to investigate the effects of elevated donor HbA1c levels (type 2 diabetes, T2D) on the islet yield and functionality postisolation. In this retrospective analysis, donors for islet isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) and normal group (HbA1c < 6.5%, n = 308). Optimum pancreas digestion time (switch time) was significantly higher in the T2D group compared to the normal group (13.7 ± 1.2 vs. 11.7 ± 0.1 min, respectively, p = 0.005). Islet yields were significantly lower in the T2D group compared to the control (T2D vs. control): islet equivalent (IEQ)/g (prepurification 2,318 ± 195 vs. 3,713 ± 114, p = 0.003; postpurification 1,735 ± 175 vs. 2,663 ± 89, p = 0.013) and islet particle number (IPN)/g (prepurification, 2,519 ± 336 vs. 4,433 ± 143, p = 0.001; postpurification, 1,760 ± 229 vs. 2,715 ± 85, p = 0.007). Islets from T2D pancreata had significantly lower viability (T2D vs. control: 91.9 ± 1.6 vs. 94.4 ± 0.3%, p = 0.004) and decreased oxygen consumption rate (DOCR) (T2D vs. control: 0.09 ± 0.01 and 0.21 ± 0.03 nmol O2 100 islets−1 min−1, p = 0.049). The islets isolated from T2D donor pancreata reversed diabetes in NOD-SCID mice in 9% (2/22) compared to islets from control donor pancreata, which reversed diabetes in 67% (175/260, p < 0.001). In conclusion, this study demonstrates that elevated HbA1c (≥6.5%) is associated with impairment of islet function and lower islet yield; however, these islets could not be suitable for clinical applications.

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

The definitive measure of β-cell quality in an islet is the measurement of β-cell function, i.e., the ability of the islets to release insulin in a controlled manner in response to minute changes in ambient glucose levels. Continuous flow or dynamic perifusion of the solution containing glucose and secretagogues through the islets is the most accurate assessment of regulated insulin release in vitro. Here, we describe in detail a low cost, mini-perifusion system that can be adapted to any laboratory to assess islet function by examining dynamic insulin release in response to elevated glucose concentrations and addition of secretagogues. Human islets with purity >80% and viability >90% were perifused with low glucose (1 mM) and subsequently challenged with high glucose (16.8 mM ± KCl, 25 mM). A prototypical biphasic response to elevated glucose concentrations was observed with an average 8-fold (above basal) increase in insulin concentration at peak values. Similarly, perifusion with carbachol or exendin-4 (Byetta) with glucose (6 mM) resulted in 1.32- and 1.35-fold increase in insulin secretion above basal. Islets could be maintained in the perifusion apparatus and continued to respond to glucose for up to 3 h. At minimal financial cost and technical expertise, this apparatus can be set-up in any biological laboratory to evaluate regulated hormone release from many cell types in less than 6 h. This will allow other laboratories to measure insulin responses to their drug or modifier of interest in vitro, in a manner that better approximates islet function in vivo.