Islet Isolation

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The long noncoding RNA MALAT1 predicts human islet isolation quality

The long noncoding RNA MALAT1 predicts human islet isolation quality

Type 1 diabetes (T1D) is currently managed via multiple daily insulin injections or continuous glucose mon- itors. Transplantation of human cadaveric islets is the only approved/health care–supported cell therapy for T1D in countries, such as Australia, Canada, France, Italy, Switzerland, and the United Kingdom. In the United States, although islet autotransplants (in chronic pancreatitis) are reimbursable, islet allotransplanta- tion in individuals with T1D is performed only through clinical trials. Usually, costs of islet isolation from a human cadaveric pancreas are very high (~US$40,000/cadaveric pancreas) because of good manufacturing practice–grade (GMP-grade) reagents and workflows (1) necessary for clinical islet transplantation. Donor characteristics are important in selecting cadaveric pancreas for GMP-grade islet isolation procedures (2). Although some studies (3, 4) demonstrated donor BMI as a positive predictor of islet yield, islet viability, and insulin secretion, the Collaborative Islet Transplant Registry (CITR) data failed to validate these obser- vations (5). Although older donors tend to yield higher numbers of islets than younger donors (6, 7), islet function appears to deteriorate with age (8). Therefore, islet isolation centers often face the difficult ques- tion of whether cost- and resource-intensive GMP-grade ($40,000) or the less pricey (~$700) research-grade reagents should be used for an available donor cadaveric pancreas. There is a need to identify biomarkers that can predict the quality and not just higher numbers of islets within donor pancreases, before under- taking this resource-, time-, and cost-intensive islet isolation procedure. Such a biomarker should not only
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A Practical Guide to Rodent Islet Isolation and Assessment

A Practical Guide to Rodent Islet Isolation and Assessment

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glu- cose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also in- cluded that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully pro- curing and purifying high-quality islets for research purposes.
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Optimization of pancreatic islet isolation from rat and evaluation of islet protective potential of a saponin isolated from fruits of Momordica dioica

Optimization of pancreatic islet isolation from rat and evaluation of islet protective potential of a saponin isolated from fruits of Momordica dioica

of islets requires enzymatic and mechanical digestion of the exocrine tissue, and density gradient separation results in the isolation of 200–400 islets. Similarly, Sheng et al. (2009) reported a classical procedure that includes three steps: collagenase perfusion, pancreas digestion, and islet purification. The whole procedure takes 30–45 minutes for each individual, and a reasonable number of islets can be obtained in a relatively short period of time. Islet beta-cell replacement and regeneration are the keys approached for the treatment of diabetic patient. The current research has provided the proof of principle that the islet isolation was executed. The pancreas was removed and washed with HBSS, and the superficial fatty tissues and blood clots were removed. The most optimum method was found to be the pancreas mincing and collagenase type XI digestion followed by filtration gradient centrifugation and cell straining, and after density gradient centrifugation, the islets obtained were passed through a prewetted, inverted polypropylene 70-µm cell strainer. The islets were incubated in RPMI at 37°C in 5% CO 2 for 48 hours, handpicked postincubation, and yielded the best islets both in terms of the quality and quantity.
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Introducing a New Experimental Islet Transplantation  Model using Biomimetic Hydrogel and a Simple  High Yield Islet Isolation Technique

Introducing a New Experimental Islet Transplantation Model using Biomimetic Hydrogel and a Simple High Yield Islet Isolation Technique

In the conventional islet isolation techniques, large amounts of enzyme and multiple toxic density gradients of histopaque are needed. Furthermore, in situ pancreas perfusion through common bile duct or other applicable sites is costly and complicated and requires extensive expertise [17,22,32,33] . Some optimal gradient components, including Ficoll, Ficoll-based histopaque, dextran, and iodixanol are commonly used for purifying islets [34-36] . Li et al. [37] used about 5-ml collagenase type XI to digest every pancreas and injected it through common bile duct by clumping the ampulla on the duodenum wall in a microscopic manner. Similar to Carter et al. [32] who used three gradients of 1.109, 1.096, and 1.070, Wilson and co- workers [38] followed the digestion of purified islets by three discontinuous gradients of histopaque, including 1.108, 1.096, and 1.037. As a matter of fact, the most challenging technical step in conventional procedures is in situ pancreas perfusion or cannulation of the common bile duct.
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Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by Clostridium histolyticum

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by Clostridium histolyticum

In this study we systematically studied the activity of a new collagenase product which was being developed by MB Pharma (Prague, Czech Republic). Sequenc- ing of bacterial genomes revealed collagenase genes duplicity in two strains (MB 201 and MB 202), while in the strain MB 204 deletion of one copy of collagenase gene was confirmed. For production, the strain with only a single collagenase gene set was selected because of its high collagenase production rate, probably due to a stronger promoter activity. Collagenase from the selected strain was used for pancreatic islet isolation and the parallel results were used as a feed-back for modification of the culture and purification method in order to achieve stable results in terms of the islet isolation outcome. The final product now can be recommended for islet isolation in a rat model.
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Robot assisted pancreatoduodenectomy with preservation of the vascular supply for autologous islet cell isolation and transplantation: a case report

Robot assisted pancreatoduodenectomy with preservation of the vascular supply for autologous islet cell isolation and transplantation: a case report

In addition to providing a platform to perform a mini- mally invasive pancreatectomy in the technically demanding setting of CP, robot assistance offers the possibility to preserve the vascular supply of the pan- creatic head during surgery for islet isolation. In consid- eration of the high rate of diabetes (approximately 50% five years after onset) observed in the natural history of CP [11,12], surgical intervention that successfully pro- vides pain relief and preserves the endocrine function earlier in the course of CP would be of significant bene- fit to the patient. Herein we present a case report docu- menting our experience with robot-assisted pylorus- preserving pancreatoduodenectomy (RA-PPPD) with minimal warm ischemia time and excellent islet recov- ery for AIT in a patient with CP.
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Clathrin-mediated Endocytosis of Alpha-1 Antitrypsin is Essential for its Protective Function in Islet Cell Survival

Clathrin-mediated Endocytosis of Alpha-1 Antitrypsin is Essential for its Protective Function in Islet Cell Survival

Pro-inflammatory cytokines play critical roles in the pathogenesis of T1D as well as in the inflammatory reactions associated with islet isolation and islet transplantation. AAT is an anti-inflammatory glycoprotein that exerts beneficial effects in islet transplantation and T1D. Despite growing evidence for the anti-inflammatory properties demonstrated for AAT, the mechanism of a direct effect of AAT on β cells remains to be defined. In this study, we found that AAT is internalized by β cells through clathrin-dependent endocytosis, leading to the suppression of the JNK pathway, inhibition of caspase 9 activation, and inhibition of mitochondria- mediated apoptosis. Furthermore, the benefit of AAT internalization was translated in vivo where we observed improved islet graft survival after syngeneic islet transplantation in mice receiving islets isolated from AAT treated donors. These results provide new insights into the mechanism(s) by which AAT achieves its protective effect on cytokine-induced islet damage.
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Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges

Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges

that an obese donor is a good candidate for successful islet isolation and transplantation. To date, “optimal” pancreata are allocated for whole organ transplantation in most cen- ters, as this procedure has historically established success in single-donor transplant scenarios. This procedure is not without inherent perioperative risks. Supporting this notion is a recent report by Berney and Johnson who conclude that the islet mass transplanted does not unequivocally correlate with islet graft function. Further arguing that based on these premises donor selection criteria for islet transplantation and hence allocation rules (pancreas for whole organ or islet transplant) may need to be redefined. 33
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Pancreatic islet transplantation

Pancreatic islet transplantation

Considering the negative impact of brittle diabetes on the quality of life (QoL), especially in those patients experi- encing severe hypoglycemic episodes, QoL analyses is an important endpoint of clinical trials. While some authors demonstrated that islet transplantation significantly reduces fear of hypoglycemia but have little impact on health-related overall QoL [20], others identified a signif- icant improvement with the use of a diabetes-specific questionnaire (DQoL). In this latter study, the most com- mon reported beneficial effects were glycemic stability and absence of hypoglycemia resulting in a feeling of independence not experienced before the transplant [21]. Difficulties Related to Pancreatic Islet Transplantation In addition to the scarcity of organs available for trans- plantation, pancreatic islets transplantation still faces major challenges, specially those related to cell loss during the process of islet isolation and the losses related to the graft site, apoptosis, allorejection, immunosuppression, and autoimmunity. The main strategies to optimize pan- creatic islet transplantation aim at improving all these aspects.
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Islet alloautotransplantation: Allogeneic pancreas transplantation followed by transplant pancreatectomy and islet transplantation

Islet alloautotransplantation: Allogeneic pancreas transplantation followed by transplant pancreatectomy and islet transplantation

Here we report a successful islet transplantation with islets isolated from a pancreas allograft that had been removed in an emergency setting due to life-threatening bleeding from a mycotic aneurysm of the Y-graft. Subsequent islet isolation and transplantation resulted in excellent endogenous insulin production, with low insulin requirement and a near-normal glucose tolerance and HbA1c. This corresponded with a high C-peptide response to a mixed meal tolerance test. We term this procedure islet allo-autotransplantation to indicate the transplantation of islets
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Improving the use of donor organs in pancreas and islet of Langerhans transplantation

Improving the use of donor organs in pancreas and islet of Langerhans transplantation

Transplantation of islets of Langerhans can improve metabolic control and quality of life in patients with longstanding type 1 diabetes (1-4). Sufficient islet numbers can be obtained from a single donor, but generally more than one islet preparation per recipient is required to obtain insulin independence (1, 3, 5-7). An important factor in determining the islet isolation outcome is the amount of endocrine tissue present in a specific pancreas. However, a high endocrine content does not ensure a high isolation yield. Other factors, such as collagen and other matrix elements are thought to play a role (8-15). When studying histological characteristics of the human pancreas in relation to islet isolation, a remarkably high number of hyperemic islets (HIs) was encountered, a phenomenon that, besides our previous report in pigs (16), has not been described in detail before. The abnormalities observed in these HIs ranged from a single dilated vessel through multiple widely dilated vessels to hemorrhages extending into the surrounding exocrine tissue. In some cases, the endocrine tissue was reduced to a small rim of cells, with only a few scattered cells left, which may consequently affect isolation yield.
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Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin

Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin

Rat islet isolation was performed in accordance with the European Council Directive 2010/63/UE and the recom- mendations of the French Ministry of Agriculture (2013-118) on the care and use of laboratory animals. Procedures were approved (n°9998_LBFA-U1055) by the local ethics committee and agreed by the French Minis- try of Research. All protocols were carried out in the animal facility of the Department of Biology of the Uni- versity (D3842110001). Rat islet isolation was performed as previously described [27]. Briefly, a cannula was inserted in the rat’s Wirsung duct to perfuse with colla- genase type IX, 1 mg ml − 1 (Sigma-Aldrich, St. Louis, MO, USA). Perfused pancreas was removed and digested in a warm bath at 37 °C for 11 min. The digested pan- creas was washed three times with a solution composed of Hank’s balanced salt solution (HBSS) (Pan Biotech, Aidenbach, Germany) supplemented with 10% of foetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA). Islets were purified by Histopaque (Sigma-Al- drich, St. Louis, MO, USA) density gradient centrifuga- tion. After three additional washes by sedimentation to eliminate residual exocrine tissue, the islet pellet ob- tained is resuspended in 1 ml of RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supple- mented with 10% foetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, 100 U ml − 1 penicillin, 100 μg ml − 1 streptomycin and 1 mM sodium pyruvate (Pan Biotech, Aidenbach, Germany). One twen- tieth of this suspension is taken and seeded in a grid box containing 2 ml of medium and 200 μl of dithizone 0.4 mg ml − 1 in dimethyl sulfoxide (DMSO) solution. This solution permits to differentiate the endocrine tissue (i.e., the islets) from the exocrine tissue. Under micros- copy, the islets are counted, measured and converted into islet equivalent (IEQ). One IEQ corresponds to the tissue volume of a perfectly spherical islet with a diam- eter of 150 μm. This procedure standardizes islet volume measurements and ensures a distribution of an equiva- lent islet volume between each condition. The rest of the islet suspension was seeded. Isolated islets were in- cubated at 37 °C in a humidified atmosphere (95% air, 5% CO 2 ) for 12 h before co-culture with MSCs. Then
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Mesenchymal stem cell therapy in type 2 diabetes mellitus

Mesenchymal stem cell therapy in type 2 diabetes mellitus

by key transcription factors such as Pdx-1, Ngn-3, Neu- roD1, Pax4, and Pax6 [36]. Correct reprogramming of cells to activate these pathways is necessary for induc- ing differentiation of MSCs into IPCs. Chen et  al. first obtained incompletely differentiated IPCs expressing insulin and nestin by culturing rat BM-MSCs in serum- free medium in the presence of a high glucose concen- tration, nicotinamide, and β-mercaptoethanol [37]. Since then, modified protocols with different stimulating agents have been applied to improve the differentiation and effi- cacy; however, results in  vivo have not been encourag- ing [38, 39]. Moriscot et al. first induced differentiation of human BM-MSCs into IPCs using adenoviral vectors coding for mouse Pdx-1 and Xie induced human BM- MSCs into IPCs using a three-step differentiation proto- col (the addition of Activin A as the differentiating agent was the final step), and the resulting cells had the capac- ity to release insulin in a glucose-dependent manner [40, 41]. Chandra et  al. [35] reported the generation of IPC aggregates from murine adipose tissue-derived stem cells, and these cells yielded numerous secretory granules within the cell cytoplasm after 10-day in  vitro culture. Calcium alginate-encapsulated IPCs, when transplanted into streptozotocin-induced diabetic mice, restored normoglycemia within 2  weeks. Some researchers have recently indicated that UC-MSCs, especially Wharton’s jelly-derived MSCs (WJ-MSCs), which are easy to source and culture, can be successfully differentiated into IPCs. Some comparative studies have also demonstrated that WJ-MSCs have superior differentiation potential towards a mature beta cell phenotype as compared to BM-MSCs [42]. Chao et al. successfully differentiated WJ-MSCs into IPCs through a stepwise culture protocol using neuron- conditioned medium in vitro and proved that the differ- entiated IPCs exhibit typical beta cell functions in  vivo [43]. Tsai et  al. injected undifferentiated WJ-MSCs expressing green fluorescent protein (GFP) into non- obese diabetic (NOD) mice and observed co-localization of human C-peptide and GFP in the pancreas, indicating that WJ-MSCs differentiated into IPCs in vivo [44]. Wu et al. compared the differentiation potential of WJ-MSCs and BM-MSCs. The results showed that both stem cell types were able to form islet-like clusters in a medium containing nicotinamide, activin, hepatocyte growth fac- tor, exendin-4, and pentagastrin. The expression of Pdx- 1, insulin secretion, and mRNA expression of insulin and C-peptide in differentiated WJ-MSCs were greater rela- tive to levels in differentiated BM-MSCs [42].
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Association of insulin, C-peptide and blood lipid patterns in patients with impaired glucose regulation

Association of insulin, C-peptide and blood lipid patterns in patients with impaired glucose regulation

Fasting blood samples were taken from all participants to detect glycosylated hemoglobin (HbA1c), blood glucose (BG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low dens- ity lipoprotein cholesterol (LDL-C)) as well as insulin and C-peptide (CP). All indexes were monitored at 30 min, 60 min, 120 min and 180 min after subjects had orally taken 75 g glucose. The C-peptide is the split product of proinsulin, which is generated when the islet β cells produce an insulin molecule and measurements of insulin and C-peptide can be used to reflect the reserve functional status of islet β cells.
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Is Islet Transplantation a Realistic Therapy for the Treatment of Type 1 Diabetes in the Near Future?

Is Islet Transplantation a Realistic Therapy for the Treatment of Type 1 Diabetes in the Near Future?

This widespread implementation of the Edmonton protocol is already driv- ing the standardization of islet process- ing techniques and will further reveal the toxicity (or lack thereof) of the immunosuppressive strategy. But are the subjects of the ITN effort the most appropriate patients to receive this elab- orate and expensive therapy? Is it appro- priate to subject healthier diabetic patients to the risks of long-term immunosuppression (nephrotoxicity, infections, malignancy, teratogenicity) in an attempt to prevent end-organ dam- age from diabetes? Or, should this limit- ed resource be reserved for uremic patients in whom diabetic control is very important, 57 but who have already
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Pancreatic β- and α-cell adaptation in response to metabolic changes

Pancreatic β- and α-cell adaptation in response to metabolic changes

mg/kg BrdU subcutaneously twice daily during the final 7 days of the 6-week study period. The transplanted recipient mice received 1 mg/ml BrdU in the drinking water (refreshed every other day) during the final 7 days. Sections were double stained for insulin-LPR and BrdU (BrdU staining kit, Invitrogen). BrdU-positive β-cells were assessed as a proportion of all β-cells per pancreatic region or islet graft. Second, sections were double stained with goat anti-Ki67 IgG (Santa Cruz Biotechnology) and guinea-pig anti-insulin IgG (Millipore) overnight at 4ºC after heat-induced antigen retrieval in 0.01 M citrate buffer (pH 6.0) followed by biotin-conjugated anti-goat IgG (Dako), streptavidine-Alexa 488 (Invitrogen) and TRITC-conjugated anti-guinea-pig (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA, USA). Apoptotic beta-cells were counted after being identified by immunostaining for insulin and by the terminal deoxynucleotidyl-transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay (Roche). The investigator was blind to the experimental conditions.
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Endothelial chimerism and vascular sequestration protect pancreatic islet grafts from antibody mediated rejection

Endothelial chimerism and vascular sequestration protect pancreatic islet grafts from antibody mediated rejection

Figure 6. Vascular sequestration of DSA. (A) Fluorescently labeled DSA (HB13, green; n = 3) and IgG2A isotype control (cyan; n = 3) were infused simul- taneously i.v. into C57BL/6 RAG2 KO mice previously grafted with CBA islets. Time-lapse intravital microscopy was used to monitor the intensity of fluorescence in several ROI. Upper left: representative bright field image showing islet graft outer limit (white dotted line) and ROI localization (white dashed circles), which were positioned outside islet graft vasculature (white arrowheads). Upper right: MFI in ROI was recorded from time of mAb injection (mean ± SD). Lower rows: representative images showing vascular sequestration of DSA (upper row) and isotype control (lower row). Scale bars: 150 μm. (B) The same experiment was conducted as in A, except that histamine was locally applied on islet graft 5 minutes after beginning of recording. Groups are DSA (HB13, green; n = 3) and IgG2A isotype control (cyan; n = 3). Scale bars: 150 μm. (C) Biodistribution of i.v.-transferred iodinated HB13 (HB13- 125 I)
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Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding

Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding

synchronized, diverse mechanisms can achieve this physiologically important ‘goal state’. We elucidated the mechanisms directing neurovascular development in pancreatic islets, the normal function of which depends on dynamic intercommunication between the vascular and nervous systems. By increasing or decreasing local VEGF production during islet development and in mature islets, we showed that: (1) establishment of the intra-islet vasculature by VEGF is essential for the postnatal maturation of islet innervation; (2) intra-islet endothelial cells provide crucial signals for nerves through production of axon guidance molecules and ECM components; and (3) β cells may compensate for a lack of neuronal input via neuro-islet cell plasticity. Therefore, we propose a novel role for VEGF in coordinating neurovascular development within the pancreatic islet. In this organ, VEGF serves as the initial signal in an intra-islet signaling cascade in which endocrine cells establish the vasculature that supports islet endocrine cell development and provides a scaffold for the later ingrowth of nerve fibers to form essential autonomic nervous system connections.
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Islet stress, degradation and autoimmunity

Islet stress, degradation and autoimmunity

to increased alternative mRNA splicing events providing evidence for β -cell neo-autoantigens generation during insulitis. 4 The inflammatory milieu also impacts post-translational modification (PTM) processes: the calcium-dependent enzymes tissue transglutaminase 2 and pepti- dyl amine deaminase 5 were shown to be activated in islets and den- dritic cells leading to increase in deamidation and citrullination of islet-autoantigens, improving their potency to bind HLA molecules and their immunogenic properties. 6–10 Such modifications have been demonstrated to increase visibility of β -cells to the immune system as seen by the increased T-cell response observed after chemical induc- tion of ER stress in β -cells. 11 Recently, we demonstrated that transla-
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Heat shock induces resistance in rat pancreatic islet cells against nitric oxide, oxygen radicals and streptozotocin toxicity in vitro

Heat shock induces resistance in rat pancreatic islet cells against nitric oxide, oxygen radicals and streptozotocin toxicity in vitro

nitroprusside, to enzymatically generated reactive oxygen intermediates or to streptozotocin cell lysis occurs after 4-12 h. We report here that a heat shock at 43 degrees C for 90 min reduces cell lysis from nitric oxide (0.45 mM sodium nitroprusside) by 70%, from reactive oxygen intermediates (12 mU xanthine oxidase and 0.05 mM hypoxanthine) by 80% and from streptozotocin (1.5 mM) by 90%. Heat shock induced resistance was observed immediately after termination of the 90 min culture at 43 degrees C and correlated with enhanced expression of hsp70. The occurrence of DNA strand breaks, a major early consequence of nitric oxide, reactive oxygen intermediates, or streptozotocin action, was not suppressed by heat shock treatment. However, the depletion of NAD+, the major cause of radical induced islet cell death, was suppressed after heat shock (P < 0.01). We conclude that pancreatic islet cells can rapidly activate defence mechanisms against nitric oxide, reactive oxygen intermediates and streptozotocin by culture at 43 degrees C. Islet cell survival is due to the prevention of lethal NAD+ depletion during DNA repair, probably by slowing down poly(ADP-ribose)polymerase activation.
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