Differences between human and rodent pancreatic islets: low pyruvate carboxylase, atp citrate lyase, and pyruvate carboxylation and high glucose-stimulated acetoacetate in human pancreatic islets.

Anaplerosis, the net synthesis in mitochondria of citric acid cycle intermediates, and cataplerosis, their export to the cytosol, have been shown to be important for insulin secretion in rodent beta cells. However, human islets may be different. We observed that the enzyme activity, protein level, and relative mRNA level of the key anaplerotic enzyme pyruvate carboxylase (PC) were 80-90% lower in human pancreatic islets compared with islets of rats and mice and the rat insulinoma cell line INS-1 832/13. Activity and protein of ATP citrate lyase, which uses anaplerotic products in the cytosol, were 60-75% lower in human islets than in rodent islets or the cell line. In line with the lower PC, the percentage of glucose-derived pyruvate that entered mitochondrial metabolism via carboxylation in human islets was only 20-30% that in rat islets. This suggests human islets depend less on pyruvate carboxylation than rodent models that were used to establish the role of PC in insulin secretion. Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. Glucose-stimulated human islets released insulin similarly to rat islets but formed much more acetoacetate. ß-Hydroxybutyrate augmented insulin secretion in human islets. This information supports previous data that indicate beta cells can use a pathway involving succinyl-CoA:3-ketoacid-CoA transferase and acetoacetyl-CoA synthetase to synthesize and use acetoacetate and suggests human islets may use this pathway more than PC and citrate to form cytosolic acyl-CoAs.

Early activation of the inflammatory response in the liver of brain-dead non-human primates.

BACKGROUND: Donor brain death (BD) triggers a systemic inflammatory response that reduces organ quality and increases immunogenicity of the graft. We characterized the early innate immune response induced by BD in the liver and peripheral blood of hemodinamically stable non-human primates (NHP). METHODS: Rhesus macaques were assigned to either brain death or control group. BD was induced by inflation of a subdurally placed catheter and confirmed clinically and by cerebral angiography. Animals were monitored for 6 h after BD and managed to maintain hemodynamic stability. RESULTS: Cortisol, epinephrine, nor-epinephrine, and IL-6 levels were elevated immediately after BD induction. Neutrophils and monocytes significantly increased in circulation following BD induction, while dendritic cells were decreased at 6 h post-induction. Flow cytometry revealed increased expression of chemokine receptors CxCR1, CxCR2, CCR2, and CCR5 in peripheral blood leukocytes from NHP subjected to BD. Microarray analysis demonstrated a significant up-regulation of genes related to innate inflammatory responses, toll-like receptor signaling, stress pathways, and apoptosis/cell death in BD subjects. Conversely, pathways related to glucose, lipid, and protein metabolism were down-regulated. In addition, increased expression of SOCS3, S100A8/A9, ICAM-1, MHC class II, neutrophil accumulation, and oxidative stress markers (carboxy-methyl-lysine and hydroxynonenal) were detected by immunoblot and immunohistochemistry. CONCLUSIONS: Activation of the innate immune response after BD in association with a down-regulation of genes associated with cell metabolism pathways in the liver. These findings may provide a potential explanation for the reduced post-transplant function of organs from brain dead donors. In addition, this work suggests potential novel targets to improve donor management strategies.

IL-1beta receptor blockade protects islets against pro-inflammatory cytokine induced necrosis and apoptosis.

Pro-inflammatory cytokines (PIC) impair islet viability and function by activating inflammatory pathways that induce both necrosis and apoptosis. The aim of this study was to utilize an in vitro rat islet model to evaluate the efficacy of a clinically approved IL-1 receptor antagonist (Anakinra) in blocking PIC induced islet impairment. Isolated rat islets were cultured for 48 h +/- PIC (IL-1beta, IFNgamma, and TNFalpha) and +/-IL-1ra then assayed for cellular integrity by flow cytometry, MAPK phosphorylation by proteome array, and gene expression by RT-PCR. Nitric oxide (NO) release into the culture media was measured by Griess reaction. Islet functional potency was tested by glucose stimulated insulin secretion (GSIS) and by transplantation into streptozotocin-induced diabetic NOD.scid mice. Rat islets cultured with PIC upregulated genes for NOS2a, COX2, IL6, IL1b, TNFa, and HMOX1. IL-1ra prevented the PIC induced upregulation of all of these genes except for TNFa. Inhibition of PIC induced iNOS by NG-monomethyl-L-arginine (NMMA) only blocked the increased expression of HMOX1. IL-1ra completely abrogated the effects of PIC with respect to NO production, necrosis, apoptosis, mitochondrial dysfunction, GSIS, and in vivo potency. IL-1ra was not effective at preventing the induction of necrosis or apoptosis by exogenous NO. These data demonstrate that Anakinra is an effective agent to inhibit the activation of IL-1beta dependent inflammatory pathways in cultured rat islets and support the extension of its application to human islets in vitro and potentially as a post transplant therapy.

Flow cytometric quantification of glucose-stimulated beta-cell metabolic flux can reveal impaired islet functional potency.

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet beta-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+(i)) (Fura Red), stimulated with glucose, and analyzed on a FACS Vantage flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet beta-cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+(i). Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and beta-cell glucose responsiveness as indicators of functional potency.

Validation of large particle flow cytometry for the analysis and sorting of intact pancreatic islets.

BACKGROUND: Accurate quantification of total islet yield is an essential step prior to transplantation and for research. The standard method of manually determining an islet equivalent (IEQ) count is subjective and prone to error. We evaluated Complex Object Parametric Analyzer and Sorter (COPAS) large particle flow cytometry for the determination of islet equivalent counts and purities of islet preparations. METHODS: Initial validation of the sensitivity and accuracy of the COPAS flow cytometer was performed by analysis and sorting of uniform polystyrene microspheres with sizes similar to islets. Human and Rhesus monkey islets were stained with the zinc-specific fluorescent dye Newport Green to discriminate islet from nonislet tissue. Islet sizes were extrapolated from standard curves obtained using microspheres from which individual islet volumes were calculated. IEQ counts on six islet preparations were performed by the standard manual method and compared with results obtained by automated COPAS flow cytometry. RESULTS: The COPAS flow cytometer was highly accurate in the detection and measurement of both polystyrene microspheres and islets. IEQ counts determined by COPAS flow cytometry were consistent with manual counts although subject to error when assessing preparations with significant numbers of islets embedded within acinar tissue. Size-specific islet sorting with retention of morphology and dithizone staining was also shown using the COPAS flow cytometer. CONCLUSIONS: COPAS large particle flow cytometry provides a novel automated approach for quantification of intact islets and determination of islet equivalent yield. In addition, the ability to analyze and sort islets based upon user defined criterion opens unique avenues for experimentation.

Donor Pretreatment With IL-1 Receptor Antagonist Attenuates Inflammation and Improves Functional Potency in Islets From Brain-Dead Nonhuman Primates.

Most pancreas and islet grafts are recovered from brain-dead (BD) donors. In this study we characterized the early inflammatory response induced by brain death in pancreata and islets from nonhuman primate donors and evaluated the effect of targeted anti-inflammatory intervention in the protection of pancreatic islets prior to transplantation. BD donors were monitored for 6 h and assigned to three experimental groups: group 1: BD-untreated donors (BD-UT) (n = 7), group 2: BD + donor pretreatment with IL-1ra (n = 6), and group 3: non-BD animals serving as controls (n = 7). We observed an IL-1ra-dependent reduction in the mobilization and activation of neutrophils from bone marrow and a significantly reduced accumulation of CD68(+) leukocytes in the pancreas and islets after brain death induction. Donor treatment with IL-1ra significantly decreased chemokine mRNA expression (MCP-1, IL-8, and MIP-1a) and attenuated the activation of circulating neutrophils and intraislet macrophages as demonstrated by a reduction in intracellular IL-1ß, IL-6, MCP-1, and MIP-1α expression. As a result, IL-1ra dramatically improved viability, mitochondrial membrane polarity, and islet engraftment in mice transplanted using a minimal islet mass. These results suggest that early immunomodulation targeting inflammation in the BD donor may represent an effective therapeutic strategy to improve islet quality and function prior to transplantation.

Standardized transportation of human islets: an islet cell resource center study of more than 2,000 shipments.

Preservation of cell quality during shipment of human pancreatic islets for use in laboratory research is a crucial, but neglected, topic. Mammalian cells, including islets, have been shown to be adversely affected by temperature changes in vitro and in vivo, yet protocols that control for thermal fluctuations during cell transport are lacking. To evaluate an optimal method of shipping human islets, an initial assessment of transportation conditions was conducted using standardized materials and operating procedures in 48 shipments sent to a central location by eight pancreas-processing laboratories using a single commercial airline transporter. Optimization of preliminary conditions was conducted, and human islet quality was then evaluated in 2,338 shipments pre- and postimplementation of a finalized transportation container and standard operating procedures. The initial assessment revealed that the outside temperature ranged from a mean of -4.6 ± 10.3°C to 20.9 ± 4.8°C. Within-container temperature drops to or below 15°C occurred in 16 shipments (36%), while the temperature was found to be stabilized between 15°C and 29°C in 29 shipments (64%). Implementation of an optimized transportation container and operating procedure reduced the number of within-container temperature drops (≤ 15°C) to 13% (n = 37 of 289 winter shipments), improved the number desirably maintained between 15°C and 29°C to 86% (n = 250), but also increased the number reaching or exceeding 29°C to 1% (n = 2; overall p < 0.0001). Additionally, postreceipt quality ratings of excellent to good improved pre- versus postimplantation of the standardized protocol, adjusting for preshipment purity/viability levels (p < 0.0001). Our results show that extreme temperature fluctuations during transport of human islets, occurring when using a commercial airline transporter for long distance shipping, can be controlled using standardized containers, materials, and operating procedures. This cost-effective and pragmatic standardized protocol for the transportation of human islets can potentially be adapted for use with other mammalian cell systems and is available online at http://iidp.coh.org/sops.aspx.

A simplified approach to human islet quality assessment.

BACKGROUND: The successful treatment of patients with type 1 diabetes by islet transplantation is affected by a multitude of factors of which infusion of the highest quality tissue is essential. The current standard pretransplant quality assessments lack sensitivity, accuracy, and objectivity in the determination of islet viability and potency. We hypothesized that a multiparametric approach focused on islet cell metabolic state, mitochondrial integrity, and in vitro glucose-stimulated insulin secretion (GSIS) could provide data predictive of in vivo function. The objective of this study was to validate a novel set of islet quality assays and develop a simplified islet quality scoring system for both basic research and clinical applications. METHODS: A series of 42 human islet preparations were screened using standard and novel methods, which included determination of yield, viability by fluorescent microscopy, GSIS, percentage of islet loss in culture, quantification of adenine nucleotides, flow cytometric measurement of viability, apoptosis, and mitochondrial membrane potential (MMP). In vivo functional potency was tested by minimal model transplant in streptozotocin-induced diabetic NOD.scid mice. RESULTS: Functionally potent islet preparations showed significantly greater numbers of cells with polarized MMP, higher ATP-to-ADP ratios, and increased glucose-induced insulin secretion. The MMP, ATP-to-ADP ratio, and GSIS data were combined into a single islet scoring formula that showed more than 86% accuracy in predicting in vivo functional potency. CONCLUSIONS: Our study demonstrates that a multiparametric approach using objective assessments focused on islet cell mitochondrial integrity and in vitro function can provide data predictive of in vivo function.

Adoptive transfer of islet antigen-autoreactive T cell clones to transgenic NOD.Ea(d)mice induces diabetes indicating a lack of I-E mediated protection against activated effector T cells.

Transgenic insertion of the MHC class II Ea(d)gene in NOD mice restores I-E expression and prevents T-cell-mediated autoimmune diabetes (IDDM). The specific molecular and cellular mechanisms responsible for the diabetes resistance of transgenic NOD.Ea(d)mice remain unclear. We adoptively transferred islet antigen-specific T cell clones into NOD and transgenic NOD.Ea(d)mice to evaluate the level of protection provided by I-E expression against activated effector T cells. We have found that neither neonatal or 3-5-week-old I-E-expressing NOD.Ea(d)mice can completely inhibit the diabetogenic activities of activated islet antigen-specific T cell clones. These data indicate that Ealpha protein expression in NOD antigen presenting cells (APC) does not reduce islet autoantigen presentation in the context of I-A(g7)below the threshold required for stimulation of effector/memory diabetogenic T cells. Our results suggest that the mechanism of Ealpha protein-mediated diabetes resistance in NOD mice may be "antigen ignorance," in which the quantity of islet autoantigens presented in the context of I-A(g7)by APC is reduced below the threshold required to activate nai;ve islet antigen-specific T cells.