Porcine Islet Graft Function is Affected by Pretreatment with a Caspase-3 Inhibitor

Brandhorst, Daniel; Kumarasamy, Vidya; Maatoui, Adel; Alt, A.; Bretzel, Reinhard G.; Brandhorst, Heide

doi:10.3727/000000006783981936

Cited by 14 publications

(9 citation statements)

References 30 publications

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“…The superior glucose response of NPIs isolated and cultured according to the scalable protocol compared to the standard protocol is likely due to the increased proportion of β-cells and insulin secretory capacity of NPIs isolated using the scalable protocol over the standard protocol. This enhanced function of NPIs isolated with the scalable protocol is potentially due to decreased β-cell apoptosis as a result of the short-term addition of the general CI (4–6,8,24,25). Pretreatment of human (4) or adult porcine (5) islets with caspase 3 inhibitors for 24–48 h by other groups show significant decreases in islet viability and function both in vivo and in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…This enhanced function of NPIs isolated with the scalable protocol is potentially due to decreased β-cell apoptosis as a result of the short-term addition of the general CI (4–6,8,24,25). Pretreatment of human (4) or adult porcine (5) islets with caspase 3 inhibitors for 24–48 h by other groups show significant decreases in islet viability and function both in vivo and in vitro. The positive effect of 90-min pretreatment with Z-VAD-FMK in NPIs could be attributed to the fact that it is a general CI instead of only caspase 3 and exposure time was much shorter (25), as well as the inherent resistance of NPIs to apoptosis after hypoxia (11).…”

Section: Discussionmentioning

confidence: 99%

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Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application

2016

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One challenge that must be overcome to allow transplantation of neonatal porcine islets (NPIs) to become a clinical reality is defining a reproducible and scalable protocol for the efficient preparation of therapeutic quantities of clinical grade NPIs. In our standard protocol, we routinely isolate NPIs from a maximum of four pancreases, requiring tissue culture in 16 Petri dishes (four per pancreas) in Ham's F10 and bovine serum albumin (BSA). We have now developed a scalable and technically simpler protocol that allows us to isolate NPIs from a minimum of 12 pancreases at a time by employing automated tissue chopping, collagenase digestion in a single vessel, and tissue culture/media changes in 75% fewer Petri dishes. For culture, BSA is replaced with human serum albumin and supplemented with Z-VAD-FMK general caspase inhibitor and a protease inhibitor cocktail. The caspase inhibitor was added to the media for only the first 90 min of culture. NPIs isolated using the scalable protocol had significantly more cellular insulin recovered (56.9 ± 1.4 µg) when compared to the standard protocol (15.0 ± 0.5 µg; p < 0.05). Compared to our standard protocol, recovery of β-cells (6.0 × 10(6) ± 0.2 vs. 10.0 × 10(6) ± 0.4; p < 0.05) and islet equivalents (35,135 ± 186 vs. 41,810 ± 226; p < 0.05) was significantly higher using the scalable protocol. During a static glucose stimulation assay, the SI of islets isolated by the standard protocol were significantly lower than the scale-up protocol (4.3 ± 0.2 vs. 5.5 ± 0.1; p < 0.05). Mice transplanted with NPIs using the scalable protocol had significantly lower blood glucose levels than the mice that receiving NPIs from the standard protocol (p < 0.01) and responded significantly better to a glucose tolerance test. Based on the above findings, this improved simpler scalable protocol is a significantly more efficient means for preparing therapeutic quantities of clinical grade NPIs.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application

2016

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“…The prototype example of death signaling via the extrinsic pathway is the Fas death receptor, which instigates assembly of the death-inducing signaling complex (DISC), a multi-protein complex comprising of the cytoplasmic aspects of the Fas receptor, the adaptor protein Fas-associated death domain-containing protein (FADD) and procaspase-8 ( Figure 2) [6]. Caspase-3 is a converging point of the apoptotic pathway ( Figure 2) [6], and its peptide inhibitors have been shown to prevent islet apoptosis and improve islet graft function [7][8][9]. Caspases are cysteine-containing aspartic acid-specific proteases which exist as zymogens in the soluble cytoplasm, endoplasmic reticulum (ER), mitochondrial intermembrane space, and nuclear matrix [10].…”

Section: Introductionmentioning

confidence: 99%

Apoptosis in pancreatic β-islet cells in Type 2 diabetes

Tomita

2016

Bosn J of Basic Med Sci

160

116

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Apoptosis plays important roles in the pathophysiology of Type 2 diabetes mellitus (T2DM). The etiology of T2DM is multifactorial, including obesity-associated insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. β-cell apoptosis is mediated through a milliard of caspase family cascade machinery in T2DM. The glucose-induced insulin secretion is the principle pathophysiology of diabetes and insufficient insulin secretion results in chronic hyperglycemia, diabetes. Recently, hyperglycemia-induced β-cell apoptosis has been extensively studied on the balance of pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and anti-apoptotic Bcl family (Bcl-2 and Bcl-xL) toward apoptosis in vitro isolated islets and insulinoma cell culture. Apoptosis can only occur when the concentration of pro-apoptotic Bcl-2 exceeds that of anti-apoptotic proteins at the mitochondrial membrane of the intrinsic pathway. A bulk of recent research on hyperglycemia-induced apoptosis on β-cells unveiled complex details on glucose toxicity on β-cells in molecular levels coupled with cell membrane potential by adenosine triphosphate generation through K+ channel closure, opening Ca2+ channel and plasma membrane depolarization. Furthermore, animal models using knockout mice will shed light on the basic understanding of the pathophysiology of diabetes as a glucose metabolic disease complex, on the balance of anti-apoptotic Bcl family and pro-apoptotic genes. The cumulative knowledge will provide a better understanding of glucose metabolism at a molecular level and will lead to eventual prevention and therapeutic application for T2DM with improving medications.

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“…It has been reported that more than half of the islets were lost in the initial days after syngeneic transplantation; and both apoptosis and necrosis contributed to this early β-cell death (2,3). Preserving islets after transplant is complex issues involving many factors, including mechanical damage induce by isolation process, hypoxia of transplanted islets, and the inflammatory and metabolic condition of the recipients.…”

Section: Discussionmentioning

confidence: 99%

Targeting Uncoupling Protein-2 Improves Islet Graft Function

Zhang

Shen

Mikita³

et al. 2011

Cell Transplant

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Preserving and enhancing the primary function of transplanted islets is not only crucial for improving the outcome of the islet transplantation, but is also important for reducing the islet mass required to achieve insulin independence. Uncoupling protein 2 (UCP2) is a member of the uncoupling protein family, which is localized to the inner mitochondrial membrane and negatively regulates insulin secretion in the pancreatic β-cells. In this study, we assessed the importance of UCP2 in improving islet graft primary function by using UCP2 gene-knockout (UCP2-KO) mice in a syngeneic islet transplantation model. Islets were isolated from UCP2-KO or wild-type (WT) C57BL/6J mice. The effects of deficiency of UCP2 on islet transplantation and islet function were determined. Two hundred islets from UCP2-KO, but not from WT, donors were capable of completely restoring normoglycemia in 1 week in all syngeneic diabetic recipients. Islets harvested from UCP2-KO mice secreted onefold more insulin in GSIS assay than that from WT mice, and maintained normal GSIS after 72-h exposure to high glucose challenge. In addition, UCP2-KO islets expressed twohold higher Bcl-2 mRNA than that from WT islets, and were resistant to high glucose and proinflammatory cytokine induced death. Our study explored a potential mechanism that may explain the benefit of UCP2-KO islets in islet transplantation. Targeting UCP2 may provide a novel strategy to improve primary function of transplanted islets and reduce the number of islets required in transplantation.Key words: Uncoupling protein 2 (UCP2); Diabetes; Islets transplant; Primary function INTRODUCTIONTransplanted islets are particularly vulnerable in the immediate posttransplantation period (7). Recent study indicated recipient hyperglycemia rendered islet grafts Diabetes mellitus is now fast emerging as one of the biggest health catastrophes in modern society. It was essusceptible to dysfunction and failure. An increased incidence of primary nonfunction was observed when a timated that approximately 160 million people worldwide suffered from diabetes in 2000, and this number marginal number of islets were transplanted into severely diabetic mice, in comparison with moderately diwas projected to increase to 221 million in 2010 and to 366 million in 2030 (25). abetic mice (12). Hyperglycemia increased oxidative stress and deteriorated β-cell function in transplanted isRecent progress in the pancreas' enzymatic digestion process along with novel immunosuppression strategies lets. Islet graft response to transplantation injury includes upregulation of protective as well as apoptotic has led to successful clinical trials of islet transplantation in humans (18). However, successful islet transplangenes (17). Therefore, preserving and enhancing the primary function of transplanted islets are not only crucial tation depends on the infusion of higher mean islet mass (>10,000 IE/kg) prepared from 2-4 donor pancreases to for improving the outcome of the islet transplantation, but also important for reduc...

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Porcine Islet Graft Function is Affected by Pretreatment with a Caspase-3 Inhibitor

Cited by 14 publications

References 30 publications

Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application

Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application

Apoptosis in pancreatic β-islet cells in Type 2 diabetes

Targeting Uncoupling Protein-2 Improves Islet Graft Function

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