Correction to: trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation.

[This corrects the article DOI: 10.1007/s40200-020-00519-y.].

Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells.

BACKGROUND: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP. METHODS: We evaluated concentration-dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real-time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs. RESULTS: We observed concentration-dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p-LKB1 (an oxidative stress-sensitive upstream kinase regulating AMPKα) and CPT1A (involved in ß-oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration-dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p-eIF2α, and CHOP along with activation of p-JNK1/2, p-ERK1/2, and p-P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs. CONCLUSIONS: EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.

Activation of AMP-activated protein kinase attenuates ethanol-induced ER/oxidative stress and lipid phenotype in human pancreatic acinar cells.

Primary toxicity targets of alcohol and its metabolites in the pancreas are cellular energetics and endoplasmic reticulum (ER). Therefore, the role of AMP-Activated Protein Kinase (AMPKα) in amelioration of ethanol (EtOH)-induced pancreatic acinar cell injury including ER/oxidative stress, inflammatory responses, the formation of fatty acid ethyl esters (FAEEs) and mitochondrial bioenergetics were determined in human pancreatic acinar cells (hPACs) and AR42J cells incubated with/without AMPKα activator [5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)]. EtOH treated hPACs showed concentration and time-dependent increases for FAEEs and inactivation of AMPKα, along with the upregulation of ACC1 and FAS (key lipogenic proteins) and downregulation of CPT1A (involved ß-oxidation of fatty acids). These cells also showed significant ER stress as evidenced by the increased expression for GRP78, IRE1α, and PERK/CHOP arm of unfolded protein response promoting apoptosis and activating p-JNK1/2 and p-ERK1/2 with increased secretion of cytokines. AR42J cells treated with EtOH showed increased oxidative stress, impaired mitochondrial biogenesis, and decreased ATP production rate. However, AMPKα activation by AICAR attenuated EtOH-induced ER/oxidative stress, lipogenesis, and inflammatory responses as well as the formation of FAEEs and restored mitochondrial function in hPACs as well as AR42J cells. Therefore, it is likely that EtOH-induced inactivation of AMPKα plays a crucial role in acinar cell injury leading to pancreatitis. Findings from this study also suggest that EtOH-induced inactivation of AMPKα is closely related to ER/oxidative stress and synthesis of FAEEs, as activation of AMPKα by AICAR attenuates formation of FAEEs, ER/oxidative stress and lipogenesis, and improves inflammatory responses and mitochondrial bioenergetics.

Trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation.

PURPOSE: Human islet isolation requires a defined collagenase-protease enzyme combination for obtaining a successful islet yield. While different islet laboratories use different enzyme combinations, a systematic methodology to identify optimal enzyme combinations and their concentrations within a single donor pancreas has not been tested. In this study, we designed a trisected pancreas model to test efficacy of three clinical grade enzyme blends (VitaCyte, Roche, SERVA) within a single pancreas. METHODS: Islet isolations were performed using brain-dead donor pancreases (n = 15) applying the enzyme-related design of experiments (DOEs) and the trisected model approach. After trimming, split each pancreas into three individual lobes (head, body, tail). As per the DOEs, the lobes were altered between different experiments, to minimize anatomical bias. Islets isolated from each lobe (27 lobes totally) were subjected to functional assessments. Insulin staining and islet area fraction were determined for tissue sections obtained from each lobe. RESULTS: Utilizing the trisected model, we identified that the collagenase dose from three different vendors did not affect the pancreas digestion and islet yield, but islet morphology after isolation with the neutral protease and BP-protease was better than thermolysin. In addition, the head lobe yielded a lower islet mass and higher tissue volume compared to other two lobes, irrespective of enzyme combination used. CONCLUSIONS: This study demonstrates that the trisected model is a promising methodology in assessing donor and isolation associated parameters. Based on this study, we conclude that the donor characteristics and an optimal enzyme dose play a critical role in achieving higher islet yields.

Human pancreatic tissue dissociation enzymes for islet isolation: Advances and clinical perspectives.

BACKGROUND AND AIMS: Successful clinical human allo or auto-islet transplantation requires the recovery of a sufficient number of functional islets from either brain-dead or chronic pancreatitis pancreases respectively. METHODS: In the last two decades (2000-2019), significant progress has been made in improving the human islet isolation procedures and in standardizing the use of different tissue dissociation enzyme (TDE; a mixture of collagenase and protease enzymes) blends to recover higher islet yields. RESULTS AND CONCLUSIONS: This review presents information focusing on properties and role of TDE blends during the islet isolation process, particularly emphasizing on the current developments, associated challenges and future perspectives within the field.

Ductal Cell Reprogramming to Insulin-Producing Beta-Like Cells as a Potential Beta Cell Replacement Source for Chronic Pancreatitis.

Islet cell auto-transplantation is a novel strategy for maintaining blood glucose levels and improving the quality of life in patients with chronic pancreatitis (CP). Despite the many recent advances associated with this therapy, obtaining a good yield of islet infusate still remains a pressing challenge. Reprogramming technology, by making use of the pancreatic exocrine compartment, can open the possibility of generating novel insulin-producing cells. Several lineage-tracing studies present evidence that exocrine cells undergo dedifferentiation into a progenitor-like state from which they can be manipulated to form insulin-producing cells. This review will present an overview of recent reports that demonstrate the potential of utilizing pancreatic ductal cells (PDCs) for reprogramming into insulin- producing cells, focusing on the recent advances and the conflicting views. A large pool of ductal cells is released along with islets during the human islet isolation process, but these cells are separated from the pure islets during the purification process. By identifying and improving existing ductal cell culture methods and developing a better understanding of mechanisms by which these cells can be manipulated to form hormone-producing islet-like cells, PDCs could prove to be a strong clinical tool in providing an alternative beta cell source, thus helping CP patients maintain their long-term glucose levels.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

Endothelial cell regulation through epigenetic mechanisms: Depicting parallels and its clinical application within an intra-islet microenvironment.

The intra-islet endothelial cells (ECs), the building blocks of islet microvasculature, govern a number of cellular and pathophysiological processes associated with the pancreatic tissue. These cells are key to the angiogenic process and essential for islet revascularization after transplantation. Understanding fundamental mechanisms by which ECs regulate the angiogenic process is important as these cells maintain and regulate the intra-islet environment facilitated by a complex signaling crosstalk with the surrounding endocrine cells. In recent years, many studies have demonstrated the impact of epigenetic regulation on islet cell development and function. This review will present an overview of the reports involving endothelial epigenetic mechanisms particularly focusing on histone modifications which have been identified to play a critical role in governing EC functions by modifying the chromatin structure. A better understanding of epigenetic mechanisms by which these cells regulate gene expression and function to orchestrate cellular physiology and pathology is likely to offer improved insights on the functioning and regulation of an intra-islet endothelial microvascular environment.

Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield.

A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM-1200 WU rC2 and 10 million collagen-degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100-g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100-g pancreas. Low-dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low-dose NCEM (12WU/g). Additionally, low-dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 µU/mL; P < .05) compared with standard-dose NCEM. Nude mouse bioassay demonstrated better islet function for low-dose RCEM (area under the curve [AUC] 24 968) compared with low-dose (AUC-38 225) or standard-dose NCEM (AUC-38 685), P < .05. This is the first report indicating that islet function can be improved by using low-dose rC1rC2 (RCEM).

Pig islet xenotransplantation.

PURPOSE OF REVIEW: The current article reviews the rationale, sources and preparation of pig islets for xenotransplantation, and presents current progress in solving the problems associated with establishing pig islet transplant as a clinical treatment for type 1 diabetes. SUMMARY: Islet transplantation is an effective treatment option for type 1 diabetes, but the available supply of human pancreases is insufficient to meet the need and demand for obtaining islets. Pig islets provide a readily available source for islet transplantation, with trials in non-human primates demonstrating their potential to reverse diabetes. The risk of zoonosis can be reduced by designated pathogen-free breeding of the donor pigs, but porcine endogenous retroviruses (PERVs) that are integrated into the genome of all pigs are especially difficult to eliminate. However, clinical trials have demonstrated an absence of PERV transmission with a significant reduction in the number of severe hypoglycemic episodes and up to 30% reduction in exogenous insulin doses. A number of methods such as production of various transgenic pigs to better xenotransplantation efficiency and the encapsulation of islets to isolate them from the host immune system are currently being tested to overcome the xenograft immune rejection. Furthermore, ongoing research is also shedding light on factors such as the age and breed of the donor pig to determine the optimal islet quantity and function.

Intra-islet endothelial cell and ß-cell crosstalk: Implication for islet cell transplantation.

The intra-islet microvasculature is a critical interface between the blood and islet endocrine cells governing a number of cellular and pathophysiological processes associated with the pancreatic tissue. A growing body of evidence indicates a strong functional and physical interdependency of ß-cells with endothelial cells (ECs), the building blocks of islet microvasculature. Intra-islet ECs, actively regulate vascular permeability and appear to play a role in fine-tuning blood glucose sensing and regulation. These cells also tend to behave as "guardians", controlling the expression and movement of a number of important immune mediators, thereby strongly contributing to the physiology of islets. This review will focus on the molecular signalling and crosstalk between the intra-islet ECs and ß-cells and how their relationship can be a potential target for intervention strategies in islet pathology and islet transplantation.

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

UNLABELLED: Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. METHODS: We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). RESULTS: Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. CONCLUSIONS: A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

A Multicenter Study: North American Islet Donor Score in Donor Pancreas Selection for Human Islet Isolation for Transplantation.

Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents). With the aid of univariate logistic regression analyses, we developed the North American Islet Donor Score (NAIDS) ranging from 0 to 100 points. The c index in the development cohort was 0.73 (95% confidence interval 0.70-0.76). The success rate increased proportionally as the NAIDS increased, from 6.8% success in the NAIDS < 50 points to 53.7% success in the NAIDS ≥ 80 points. We further validated the NAIDS using a separate set of data consisting of 179 islet isolations. A comparable outcome of the NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in clinical practice. Apart from its utility in clinical decision making, the NAIDS may also be used in a research setting as a standardized measurement of pancreas quality.

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 µM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

BACKGROUND: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity. METHODS: Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis. RESULTS: Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72). CONCLUSIONS: Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

Factors affecting transplant outcomes in diabetic nude mice receiving human, porcine, and nonhuman primate islets: analysis of 335 transplantations.

BACKGROUND: In the absence of a reliable islet potency assay, nude mice (NM) transplantation is the criterion standard to assess islet quality for clinical transplantation. There are factors other than islet quality that affect the transplant outcome. METHODS: Here, we analyzed the transplant outcomes in 335 NM receiving islets from human (n=103), porcine (n=205), and nonhuman primate (NHP; n=27) donors. The islets (750, 1000, and 2000 islet equivalents [IEQ]) were transplanted under the kidney capsule of streptozotocin-induced diabetic NM. RESULTS: The proportion of mice that achieved normoglycemia was significantly higher in the group implanted with 2000 IEQ of human, porcine, or NHP islets (75% normoglycemic) versus groups that were implanted with 750 IEQ (7% normoglycemic) and 1000 IEQ (30% normoglycemic). In this study, we observed that the purity of porcine islet preparations (P≤0.001), islet pellet size in porcine preparations (P≤ 0.01), and mice recipient body weight for human islet preparations (P=0.013) were independently associated with successful transplant outcome. NHP islets of 1000 IEQ were sufficient to achieve normoglycemic condition (83%). An islet mass of 2000 IEQ, high islet purity, increased recipient body weight, and high islet pellet volume increased the likelihood of successful reversal of diabetes in transplanted mice. Also, higher insulin secretory status of islets at basal stimulus was associated with a reduced mouse cure rate. The cumulative incidence of graft failure was significantly greater in human islets (56.12%) compared with porcine islets (35.57%; P≤0.001). CONCLUSION: Factors affecting NM bioassay were identified (islet mass, islet purity, pellet size, in vitro insulin secretory capability, and mouse recipient body weight) and should be considered when evaluating islet function.

A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products.

BACKGROUND: The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). METHODS: We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. RESULTS: The NEM consistently achieved higher islet yields from pancreatitis (P<0.003) and deceased donor pancreases (P<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510 ± 2150 islet equivalent number/gram (IEQ/g) pancreas and 694,681 ± 147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 200,000 IEQ from 19 of 21 pancreases (averaging 422,893 ± 181,329 total IEQ and 5979 ± 1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. CONCLUSIONS: A NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.

Antiapoptotic actions of exendin-4 against hypoxia and cytokines are augmented by CREB.

Islets isolated from cadaveric donor pancreas are functionally viable and can be transplanted in diabetic patients to reduce insulin requirements. This therapeutic approach is less efficient because a significant portion of functional islets is lost due to oxidative stress, inflammation, and hypoxia. Exendin-4, a glucagon-like peptide-1 receptor agonist, is known to improve islet survival through activation of the transcription factor, cAMP response element binding protein (CREB). However, isolated human islets are exposed to several stresses known to down-regulate CREB. The objective of the present study was to determine whether the cytoprotective actions of exendin-4 in human islets can be augmented by increasing the levels of CREB. Simulation of ischemia/reperfusion injury and exposure to hypoxic conditions in cultured human islets resulted in decreased CREB activation and induction of apoptosis. Islets were transduced with adenoviral CREB followed by exposure to exendin-4 as a strategy for improving their survival. This combination increased the levels of several proteins needed for ß-cell survival and function, including insulin receptor substrate-2, Bcl-2, and baculoviral IAP repeat-containing 3, and suppressed the expression of proapoptotic and inflammatory genes. A combination of CREB and exendin-4 exerted enhanced antiapoptotic action in cultured islets against hypoxia and cytokines. More significantly, transplantation of human islets transduced with adenoviral CREB and treated with exendin-4 showed improved glycemic control over a 30-d period in diabetic athymic nude mice. These observations have significant implications in the therapeutic potential of exendin-4 and CREB in the islet transplantation setting as well as in preserving ß-cell mass of diabetic patients.

Insulin degradation by acinar cell proteases creates a dysfunctional environment for human islets before/after transplantation: benefits of α-1 antitrypsin treatment.

BACKGROUND: Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of α-1 antitrypsin (A1AT). METHODS: Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations. The effect of proteases on ultrastructure of islets and ß-cell insulin granules were examined by transmission electron microscopy. The number of insulin granules and insulin-labeled immunogold particles were counted. The in vivo effect of proteases on islet function was studied by transplanting acinar cells adjacent to islet grafts in diabetic mice. The effects of A1AT culture supplementation on protease activity, insulin levels, and islet function were assessed in pure and impure islets. RESULTS: Islet loss after culture was significantly higher in impure relative to pure preparations (30% vs. 14%, P<0.04). Lower islet purity was associated with increased protease activity and decreased insulin levels in culture supernatants. Reduced ß-cell insulin granules and insulin degradation by proteases were confirmed by transmission electron microscopy. Transplantations in mice showed delayed islet graft function when acinar cells were transplanted adjacent to the islets under the kidney capsule. Supplementation of A1AT to impure islet cultures maintained islet cell mass, restored insulin levels, and preserved islet functional integrity. CONCLUSION: Culture of impure human islet fractions in the presence of A1AT prevents insulin degradation and improves islet recovery.