SummaryThe T lymphocytes mediating autoimmune destruction of pancreatic [3 cells in the nonobese diabetic (NOD) mouse model of insulin-dependent diabetes mellitus (IDDM) may be generated due to functional defects in hematopoietically derived antigen-presenting cells (APC). However, it has not been clear which particular subpopulations ofAPC (B lymphocytes, macrophages, and dendritic cells) contribute to the development and activation of diabetogenic T cells in NOD mice. In the current study we utilized a functionally inactivated immunoglobulin (Ig)l.* allele (Ig/x ''a) to generate a "speed congenic" stock of B lymphocyte-deficient NOD mice that are fixed for linkage markers delineating previously identified diabetes suscepnbility (Ida") genes, These B lymphocyte NOD.Igi.,, ''tt mice had normal numbers of T cells but were free of overt IDDM and insulitis resistant, while the frequency of disease in the B lymphocyte intact segregants was equivalent to that of standard NOD mice in our colony. Thus, B lymphocytes play a heretofore unrecogmzed role that is essential for the initial development and/or activation of [3 cell autoreactive T cells in NOD mice.

We have developed a luminol-based assay using intact islets, which allows for quantification of reactive oxygen species (ROS). In addition, an index capable of characterizing metabolic and mitochondrial integrity prior to transplantation was created based on the capacity of islets to respond to high glucose and rotenone (mitochondrial respiratory chain complex I inhibitor) by production of ROS.To validate this assay, lipid peroxidation and antioxidative defense capacity were evaluated by detection of malondialdehyde (MDA) levels and glutathione peroxidase activity (GPx), respectively. Also, flow cytometric analyses of ROS (dihydroethidine), apoptosis (Annexin V, active caspases), necrosis (Topro3), and mitochondrial membrane potential (JC-1) were done in parallel to correlate with changes in luminol-measured ROS. ATP/ADP ratios were quantified by HPLC and the predictive value of ROS measurement on islet functional potency was correlated with capacity to reverse diabetes in a streptozotocin-induced diabetic NOD.scid mouse model as well as in human transplant recipients. Our data demonstrate that levels of ROS in islets correlate with the percentage of apoptotic cells and their functional potency in vivo. The ROS indices following glucose and rotenone exposure are indicative of metabolic potency and mitochondrial integrity and can be used as surrogate markers to evaluate the quality of islets prior to transplantation.

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. ␤-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.Understanding the enzymatic makeup of human pancreatic islets is fundamental to developing strategies for designing artificial beta cells and beta cells differentiated from stem cells as treatments for type 1 diabetes, as well as modulating beta cell metabolism for the treatment of type 2 diabetes. Until recently, most of the information about normal insulin secretion came from studies of rodent islets or clonal cell lines. Although a recent study showed human pancreatic islets respond to insulin secretagogues similarly to rodent islets (1), what is still unknown is whether the use of intracellular pathways of secretagogue metabolism is the same in human islets as in rodent islets and cell lines. During the last few years, human islet preparations from human donors have become more readily available to researchers. By studying the levels of enzymes, the functional units of metabolism, the recent abundant supply of human islets has enabled our laboratory to discover clues suggesting differences in metabolic pathways between pancreatic islets of humans and rodents that have implications for better understanding normal human beta cell physiology.Anaplerosis, the biosynthesis of citric acid cycle intermediates (2), is widely believed to be important for insulin secretion (3). Pyruvate carboxylase (PC) 2 is the key anaplerotic enzyme in this process ...

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 48h ± PIC (IL-1β, IFNγ, and TNFα 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-1β 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.